Compositions and Methods for Modulating Growth of a Genetically Modified Gut Bacterial Cell

ABSTRACT

Compositions and methods are provided for modulating growth of a genetically modified bacterial cell present in a human organ, for modulating growth of a genetically modified bacterial cell in an organ (e.g., gut), for displacing at least a portion of a population of bacterial cells in an organ, and for facilitating gut colonization by a genetically modified bacterial cell. Also provided are genetically modified bacterial cells, e.g., cells that include a heterologous carbohydrate-utilization gene or gene set that provides for the ability to utilize as a carbon source a rare carbohydrate of interest that is utilized as a carbon source by less than 50% of bacterial cells present in a human microbiome.

CROSS-REFERENCE

This application is a Continuation of U.S. application Ser. No.16/047,862, filed Jul. 27, 2018, which is a Continuation ofInternational Application No. PCT/US2017/066408, filed Dec. 14, 2017,which claims benefit of U.S. Provisional Patent Application No.62/435,048, filed Dec. 15, 2016, which applications are incorporatedherein by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under contracts DK085025and OD006515 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“STAN-1357WO_SeqList_ST25.txt” created on Nov. 27, 2017 and having asize of 732 KB. The contents of the text file are incorporated byreference herein in their entirety.

INTRODUCTION

The gut microbiota is integral to many facets of human biology. Gutbacteria produce hundreds of metabolites that accumulate in humantissue, and the composition of the gut microbiota has been linked topathogen susceptibility, immune disorders, heart disease, centralnervous system diseases, diabetes and obesity. The rules governing gutecology are largely unknown, and colonization level of an introducedbacterial strain could theoretically be influenced by a wide range offactors including competition for a variety of macro- andmicro-nutrients and the avoidance of inhibitory interactions from otherbacteria, bacteriophage and the immune system.

There is a need in the art for compositions and methods that facilitatethe ability to colonize the gut of a patient with a desired bacterialstrain, e.g., a strain that is predicted to improve the patient'shealth. Prior to this disclosure, it has not been possible to colonizethe gut of a patient with an introduced bacterial strain such that thepopulation of the introduced strain can reliably be maintained at apredictable colonization level, despite variations in diet, hostgenetics, and composition of the bacteria already present.

SUMMARY

Compositions and methods are provided for modulating growth of agenetically modified bacterial cell present in a human organ, formodulating growth of a genetically modified bacterial cell in an organ,for displacing at least a portion of a population of bacterial cells inan organ, and for facilitating gut colonization by a geneticallymodified bacterial cell.

For example, in some cases a subject method includes introducing to ahuman organ (e.g., gut) a genetically modified bacterial cell capable ofcontrolled entrenchment and/or controlled colonization. In some cases amethod of the disclosure includes administering to a human a rarecarbohydrate of interest that is utilized as a carbon source by asubject genetically modified bacterial cell present in a human organ(e.g., gut), where less than 50% (e.g., less than 40%, less than 30%,less than 20%, less than 10%, less than 5%, less than 3%, less than 2%,less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, or none;e.g., less than 30%, less than 10%, less than 3%, less than 1%, lessthan 0.3%, less than 0.1%, less than 0.03%, less than 0.01%, less than0.003%, less than 0.001%, less than 0.0001%, or none) of other bacterialcells present in the organ utilize the rare carbohydrate of interest asa carbon source. In some cases a method of the disclosure includesintroducing a subject genetically modified bacterial cell to an organ(e.g., gut) in vivo, wherein the genetically modified bacterial cellutilizes as a carbon source a rare carbohydrate of interest, where lessthan 50% (e.g., less than 40%, less than 30%, less than 20%, less than10%, less than 5%, less than 3%, less than 2%, less than 1%, less than0.5%, less than 0.2%, less than 0.1%, or none; e.g., less than 30%, lessthan 10%, less than 3%, less than 1%, less than 0.3%, less than 0.1%,less than 0.03%, less than 0.01%, less than 0.003%, less than 0.001%,less than 0.0001%, or none) of other bacterial cells present in theorgan utilize the rare carbohydrate of interest as a carbon source. Insome cases a method of the disclosure includes introducing to an organ(e.g., gut) a genetically modified bacterial cell capable of controlledentrenchment and/or controlled colonization, and displacing at least aportion of the population of bacterial cells in the organ with thegenetically modified bacterial cell.

In some cases a method of the disclosure includes (a) introducing agenetically modified bacterial cell into a gut of an individual, wherethe genetically modified bacterial cell includes a heterologouscarbohydrate-utilization gene or gene set that provides the geneticallymodified bacterial cell with the ability to use a carbohydrate ofinterest as a carbon source; and (b) administering the carbohydrate ofinterest to the individual, thereby providing the genetically modifiedbacterial cell with the carbon source.

Provided are genetically modified bacterial cells. In some embodimentssuch a cell utilizes as a carbon source a rare carbohydrate of interestthat is utilized as a carbon source by less than 50% (e.g., less than40%, less than 30%, less than 20%, less than 10%, less than 5%, lessthan 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%,less than 0.1%, or none; e.g., less than 30%, less than 10%, less than3%, less than 1%, less than 0.3%, less than 0.1%, less than 0.03%, lessthan 0.01%, less than 0.003%, less than 0.001%, less than 0.0001%, ornone) of bacterial cells present in a human microbiome (e.g., gutmicrobiome). In some cases, such a cell includes a heterologouscarbohydrate-utilization gene or gene set that provides the geneticallymodified bacterial cell with an ability to utilize as a carbon source arare carbohydrate of interest that is utilized as a carbon source byless than 50% (e.g., less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, less than 3%, less than 2%, less than 1%, lessthan 0.5%, less than 0.2%, less than 0.1%, or none; e.g., less than 30%,less than 10%, less than 3%, less than 1%, less than 0.3%, less than0.1%, less than 0.03%, less than 0.01%, less than 0.003%, less than0.001%, less than 0.0001%, or none) of bacterial cells present in ahuman microbiome (e.g., gut microbiome). In some cases, a geneticallymodified bacterial cell includes (i) a heterologous therapeutictransgene; and (ii) a carbohydrate-utilization gene or gene set thatprovides the genetically modified bacterial cell with an ability toutilize as a carbon source a rare carbohydrate of interest that isutilized as a carbon source by less than 50% (e.g., less than 40%, lessthan 30%, less than 20%, less than 10%, less than 5%, less than 3%, lessthan 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%,or none; e.g., less than 30%, less than 10%, less than 3%, less than 1%,less than 0.3%, less than 0.1%, less than 0.03%, less than 0.01%, lessthan 0.003%, less than 0.001%, less than 0.0001%, or none) of bacterialcells present in a human microbiome (e.g., gut microbiome). In somecases, a genetically modified bacterial cell includes a heterologouscarbohydrate-utilization gene or gene set that provides the geneticallymodified bacterial cell with the ability to use a rare carbohydrate ofinterest as a carbon source.

Provided are methods of genetically modifying a bacterial cell. In somesuch cases, a subject method includes (i) providing a bacterial cell;and (ii) genetically modifying the bacterial cell to utilize as a carbonsource a rare carbohydrate of interest that is utilized as a carbonsource by less than 50% (e.g., less than 40%, less than 30%, less than20%, less than 10%, less than 5%, less than 3%, less than 2%, less than1%, less than 0.5%, less than 0.2%, less than 0.1%, or none; e.g., lessthan 30%, less than 10%, less than 3%, less than 1%, less than 0.3%,less than 0.1%, less than 0.03%, less than 0.01%, less than 0.003%, lessthan 0.001%, less than 0.0001%, or none) of bacterial cells present in ahuman microbiome (e.g., gut microbiome). In some cases, a subject methodincludes (i) providing a bacterial cell that utilizes as a carbon sourcea rare carbohydrate of interest that is utilized as a carbon source byless than 50% (e.g., less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, less than 3%, less than 2%, less than 1%, lessthan 0.5%, less than 0.2%, less than 0.1%, or none; e.g., less than 30%,less than 10%, less than 3%, less than 1%, less than 0.3%, less than0.1%, less than 0.03%, less than 0.01%, less than 0.003%, less than0.001%, less than 0.0001%, or none) of bacterial cells present in ahuman microbiome (e.g., gut microbiome); and (ii) genetically modifyingthe bacterial cell to express one or more therapeutic transgenes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Glycoside hydrolase representation in the healthy Americanmicrobiota. The incidence and mean abundance of glycoside hydrolasefamilies in 87 Human Microbiome Project metagenomes are shown. Theglycoside hydrolase family GH86 includes enzymes that can break downeither porphyran or agarose, and is underrepresented in this thesesamples, with an incidence and mean abundance of 66% and 0.00015,respectively. GH32 is associated with utilization of inulin and is morehighly represented with an incidence and mean abundance of 100% and0.0049, respectively.

FIG. 2 (panels A-E). The abundance of porphyran-utilizing Bacteroidescan be modulated by the addition of porphyran. (panel A) A strain ofBacteroides ovatus capable of utilizing porphyran (NB001) was isolatedfrom the environment. (panel B) The porphyran utilizing strain reaches ahigher (P=0.02) and more predictable (P<0.01) abundance in eightdifferent complex communities in culture when porphyran is present. Theaverage of two replicates for each community is shown. (panel C)Porphyran increased abundance of porphyran utilizer in vivo. Two groupsof conventional mice, one fed a standard (std) diet and one fed apolysaccharide deficient (pd) diet, were colonized with porphyranutilizing Bacteroides, strain NB006. Porphyran was administered in thewater, and abundance of both total anaerobic culturable bacteria andNB006 in the feces were monitored. Averages and standard deviation forreplicates of four mice are shown. (panel D) Conventional mice being feda standard diet (i.e. lacking porphyran) were administered a Bacteroidesstrain that cannot utilize porphyran, NB008. After seven days, thesemice were challenged with NB006, a Bacteroides strain which can utilizeporphyran. NB006 was cleared from the mice after five days. Abundance ofNB008 and NB006 in the feces was monitored by selective plating,averages and standard deviation for replicates of three mice are shown.(panel E) Porphyran allowed for stable colonization of a porphyranutilizer into a resistant microbiota. Conventional mice wereadministered NB008, and one week later given porphyran in the watersimultaneous to receiving NB006. Porphyran was removed from the waterand then re-introduced, while abundances of both strains in the feceswere monitored via selective plating. NB006 was able to displace NB008with the addition of porphyran, and inhibit it from re-invasion on day48. Averages and standard deviation for replicates of three mice areshown.

FIG. 3. Knockout of an eight-gene operon in the putative NB001 porphyranPUL prevents growth on porphyran. Genes corresponding to homologs ofBACPLE_1692-1699 from B. plebeius were deleted from NB001. End-pointcell density measurements were taken after growth in minimal mediasupplemented with either 0.2% glucose (blue) or 0.8% nori extract(orange).

FIG. 4. Nucleotide alignment between the porphyran mobile elements in B.plebeius (Hehemann et al., Nature 464, 908-912 (2010); Hehemann et al.,Proc. Natl. Acad. Sci. U.S.A. 109, 19786-19791 (2012)) and NB001. Threetruncations of the putative porphyran polysaccharide utilization locus(PUL), shown in green, were cloned into a conjugation vector andtransferred to a naive strain to assay growth on porphyran.

FIG. 5. Map of the medium-length porphyran PUL conjugative plasmidpWD036. The medium-length porphyran PUL genes were cloned from the NB001genome and are homologs of BACPLE_1683-1706 from the B. plebeius genome(see e.g., Tables 5 and 6).

FIG. 6. The medium-length porphyran PUL is sufficient to confer growthon porphyran in B. vulgatus. A B. vulgatus strain (NB004) containingeither the small (pWD037) or medium-length (pWD036) porphyran PUL weregrown in minimal media supplemented with either 0.2% glucose (blue) or0.8% nori extract (orange). End-point cell density measurements areshown. pWD037 contains homologs of BACPLE_1688-1699. pWD036 containshomologs of BACPLE_1683-1706 (see, e.g., Tables 5 and 6).

FIG. 7 (panels A-D). Niche availability varied by microbiota and couldbe modulated by addition of a privileged nutrient. (panel A), Schematicof experimental design. Groups of mice with three different gutcommunities, mouse (RF), or human (Hum-1, Hum-2), were colonized withNB001. NB001 was tracked in feces for seven days, and mice were switchedto specialized polysaccharide chow containing either inulin (in.) orporphyran (por.). (panel B), Density of NB001 in feces in the three gutcommunities over the course of seven days (n=10) Kruskal-Wallis test,p<0.0001. (panel C), Density of NB001 in feces prior to and uponaddition of inulin in the diet (brackets/dotted lines, RF n=4, Hum-1n=5) Kruskal-Wallis test, p=0.03. (panel D), Density in NB001 in fecesprior to and upon addition of nori extract in the diet (brackets/dottedlines, RF n=5, Hum-1 n=7, Hum-2 n=6). Kruskal-Wallis test, p=0.19. Errorbars indicate s.e.m.

FIG. 8 (panels A-F). Access to a privileged nutrient mediated populationsize and overcame isogenic self-exclusion. Density of NB001 (PUL+),NB001 lacking the ability to utilize porphyran (PUL−), and totalculturable anaerobes (Total) in feces of conventional mice. Periods ofadministration of porphyran extract in drinking water indicated bybrackets/dotted lines (panels a-c: 1% w/v, panels d-f: 0.1% w/v). (panelA), Mice colonized with PUL+ and fed a MAC-deficient diet or (panel B),MAC-rich diet demonstrated toggling of strain abundance uponadministration of porphyran extract. (panel C), Mice colonized with PUL−and fed a MAC-rich diet showed no change in strain abundance withadministration of porphyran extract. (panels D-F) Mice were colonizedwith PUL− for 6 days, and challenged with PUL+ on day 6. (panel D), PUL+is excluded by PUL− in the absence of porphyran extract. (panel E), PUL+displaces PUL− with access to porphyran for five days. (panel F), PUL+and PUL− stably co-exist after a three day pulse of porphyran extract.Error bars indicate standard deviation, n=4 for all experiments.

FIG. 9 (panels A-E). Control over population size was engineered, andwas highly tunable. (panel A), Schematic of porphyran PUL from NB001aligned to the previously published B. plebeius PUL. Shown below are thedifferent minimal PULs (Long, Medium, and Short) designed and tested forability to confer growth on porphyran extract. The eight gene regiondeleted from NB001 PUL− is shown in gray (“Knockout”). (panel B), Growthcurves for natural and engineered strains on porphyran extract as solecarbon source. (panel C), B. thetaiotaomicron harboring the mediumlength PUL colonized in conventional mice demonstrates toggling uponadministration of 1% w/v porphryan extract in the drinking water(brackets/dotted lines, n=4). Error bars indicate standard deviation.(panel D), NB001 PUL+ colonized in conventional mice demonstratestunable response to porphyran extract in the drinking water(brackets/dotted lines, n=4 per group). Error bars indicate standarddeviation. (panel E), NB001 expressing GFP colonized in conventionalmice given 0.01% porphryan extract (left) or 1% porphyran extract(right). Host epithelium visualized by DAPI (nuclei, blue), epithelialborder visualized by phalloidin (F-actin, white), background microbiotaby DAPI segmented from host epithelium (bacteria, magenta), NB001 byendogenous GFP (bacteria, green). Scale bar represents 20 μm.

FIG. 10 (panels A-B). Three model background communities of gut microbeswere distinct from each other. (panel A), Principal Coordinates Analysisof weighted UniFrac distance for 16S rDNA amplicons from feces from thethree background community groups from FIG. 7 before diet switch,conventional (RF), or humanized (Hum-1 and Hum-2), n=10. (panel B),Comparison of weighted UniFrac distances within each group (Intra) oracross groups (A×B, A×C, B×C). One-way ANOVA, p<0.0001.

FIG. 11 (panels A-C). NB001 can utilize both inulin and porphyranextract as sole carbon sources for growth. NB001 demonstrates growth inminimal media with only one specified carbon source, either (panel A),glucose (doubling time=157 minutes), (panel B), inulin (doublingtime=127 minutes), or (panel C), porphyran extract (doubling time=98minutes).

FIG. 12 (panels A-B). Porphyran extract did not significantly impact thegut microbiota in the absence of a known utilizer. Weighted UniFracanalysis was performed on fecal 16S data for conventional mice colonizedwith a porphyran utilization knockout (FIG. 8c ) before addition ofporphyran extract (Pre, n=8) or after (Post, n=9). (panel A), PrincipalCoordinates Analysis (panel B), Unpaired two-tailed t-test, p=0.25

FIG. 13 (panels A-B). Primary colonizer displacement was robust andcontingent upon access to porphyran. (panel A), Conventional mice (n=7)colonized with NB001 (PUL− 1) containing an eight-gene deletionabrogating its ability to utilize porphyran (FIG. 9b ) demonstrateresistance to subsequent challenge with an isogenic knockout strain(PUL− 2) in the presence of 1% porphryan extract in the drinking water.Notably, conventionally raised mice were permissive to colonization bythis strain and other species of Bacteroides tested (B.thetaiotaomicron, B. fragilis, B. uniformis, B. vulgatus, stablecolonization range of 8*10e5-3*10e8 c.f.u. per ml feces), which differsfrom reports of tests on other conventionally raised mice, potentiallyreflecting inter-colony microbiota differences. (panel B), Mice fromFIG. 8e were challenged with the originally colonizing porphyranutilization knockout (PUL−) that was displaced by the utilizer (PUL+)and demonstrated colonization resistance to the previously displacedknockout strain.

FIG. 14 (panels A-C). Data showing that access to porphyran allowed forcrypt invasion and stable maintenance in the presence of an exclusionaryprimary colonizing strain.

DETAILED DESCRIPTION

As summarized above, compositions and methods are provided formodulating growth of a genetically modified bacterial cell present in ahuman organ, for modulating growth of a genetically modified bacterialcell in an organ (e.g., gut), for displacing at least a portion of apopulation of bacterial cells in an organ, and for facilitating gutcolonization by a genetically modified bacterial cell. Also provided aregenetically modified bacterial cells, e.g., cells that include aheterologous carbohydrate-utilization gene or gene set that provides forthe ability to utilize as a carbon source a rare carbohydrate ofinterest that is utilized as a carbon source by less than 50% ofbacterial cells present in a human microbiome.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells (e.g., a population of suchcells) and reference to “the protein” includes reference to one or moreproteins and equivalents thereof, e.g. polypeptides, known to thoseskilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods and Compositions

Provided are methods and compositions for modulating growth of agenetically modified bacterial cell present in an organ (e.g., humanorgan) of an individual. Modulation of growth can be achieved byadministering an energy and/or carbon source (e.g., a nutrient, acarbohydrate, and the like) of interest to the individual, therebyproviding the genetically modified bacterial cell with the energy and/orcarbon source. The genetically modified bacterial cell can include agene or gene set (e.g., a carbohydrate-utilization gene or gene set,e.g., in some cases a heterologous gene or gene set) that provides thegenetically modified bacterial cell with the ability to use the energyand/or carbon source. As an example, in some cases a subject geneticallymodified bacterial cell includes a heterologous gene or gene set thatprovides the genetically modified bacterial cell with the ability to usea carbohydrate of interest as a carbon source.

In some cases a subject method includes a step of introducing thegenetically modified bacterial into the organ (e.g., into a gut of anindividual) prior to the modulating growth step. Thus, in someembodiments a genetically modified bacterial cell is introduced into anindividual (e.g., into the individual's gut). The individual can be anymammalian species, e.g. rodent (e.g., mouse, rat), ungulate, cow, pig,sheep, camel, rabbit, horse, dog, cat, primate, non-human primate,human, etc. Thus, in some cases the individual is human. The individualmay be a neonate, a juvenile, or an adult. In some cases, theintroduction is by oral administration. Any convenient type of oraladministration can be used. For example, oral administration can includedelivery via eating (e.g., incorporated into food), drinking (e.g.,incorporated into a solution such as drinking water), oral gavage (e.g.,using a stomach tube), aerosol spray, tablets, capsules, pills, powders,and the like. In some embodiments, a genetically modified bacterial cellis introduced into an individual (e.g., into the individual's gut) bydelivery into the individual's colon. Any convenient number ofgenetically modified cells can be introduced. For example, in some cases10³ or more cells (e.g., 10⁴ or more, 10⁵ or more, 10⁶ or more, 10⁷ ormore, 10⁸ or more cells, 10⁹ or more, or 10¹⁰ or more) cells areintroduced. In some cases 10¹¹ or more cells are introduced. In somecases, between 10⁷ and 10¹³ cells are introduced (e.g., between10⁸-10¹², 10⁹-10¹², or 10¹⁰-10¹² cells).

i. Growth Control

Provided are methods for modulating growth of a genetically modifiedbacterial cell present in an organ (e.g., a gut) of an individual (e.g.,a human). Growth can be controlled by providing (e.g., administering tothe individual) a carbohydrate that can be utilized by the geneticallymodified bacterial cell as a carbon source. For example, growth can becontrolled by adjusting the amount of carbohydrate provided and/orfrequency with which the carbohydrate is provided.

In some cases, a privileged niche is created by using a carbon source(e.g., carbohydrate) that is uncommon in the diet of the individual andis either rarely or not consumed by the gut bacteria in the populationof interest (e.g., human population of interest). The term ‘privilegedniche’ is used herein to refer to a situation in which the geneticallymodified bacteria have ‘privileged’ access to a resource (e.g., a carbonsource), which thereby provides them with a growth advantage (at leastwith respect to that resource). The use of a carbon source (e.g., acarbohydrate such as porphyran) to create a privileged niche for anintroduced bacteria allows the bacteria to establish a population (e.g.,colonize, entrench, etc.) in the target organ (e.g., gut). Moreover,because the introduced bacteria has privileged access, the amount of thecarbon source (e.g., carbohydrate) that is made available to theintroduced bacteria can be manipulated (e.g., increased, decreased, ormaintained) to control the growth of the population of introducedbacteria (e.g., in some cases without perturbing resident bacterialpopulations). In some embodiments, this results in a situation in whichthe abundance of introduced bacteria at any given time is predictable,e.g., based on the amount of privileged resource (e.g., carbohydratesuch as porphyran) that is being provided (e.g., introduced into thediet of the individual) (see, e.g., FIG. 2, panel B). For example, insome cases the deviation in abundance of the introduced bacteria betweendifferent microbial communities by the 7th day after introduction (or7th day after introduction, where the introduced bacteria had access tothe privileged resource) spans a range of 4-fold or less (e.g., 3-foldor less).

In some embodiments, e.g., to provide a privileged niche to theintroduced genetically modified bacteria, the carbohydrate of interestis a rare carbohydrate of interest. By ‘rare’ carbohydrate of interest,it is meant a carbohydrate of interest that is utilized by less than 50%(e.g., less than 40%, less than 30%, less than 20%, less than 10%, lessthan 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, lessthan 0.2%, less than 0.1%, or none; e.g., less than 30%, less than 10%,less than 3%, less than 1%, less than 0.3%, less than 0.1%, less than0.03%, less than 0.01%, less than 0.003%, less than 0.001%, less than0.0001%, or none) of other bacterial cells present in a target organsuch as the gut (i.e., cells ‘other’ than the introduced/geneticallymodified bacteria, e.g., cells of the resident population prior tointroduction). Thus, in some cases (e.g., in any of the methods of thedisclosure), a rare carbohydrate of interest is one that can be utilizedby less than 50% (e.g., less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, less than 3%, less than 2%, less than 1%,less than 0.5%, less than 0.2%, less than 0.1%, or none; e.g., less than30%, less than 10%, less than 3%, less than 1%, less than 0.3%, lessthan 0.1%, less than 0.03%, less than 0.01%, less than 0.003%, less than0.001%, less than 0.0001%, or none) of other bacterial cells present inthe organ. In some cases (e.g., in any of the methods of thedisclosure), the rare carbohydrate of interest is one that can beutilized by less than 20% (e.g., less than 10%, less than 5%, less than3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, lessthan 0.1%, or none) of other bacterial cells present in the organ. Insome cases (e.g., in any of the methods of the disclosure), the rarecarbohydrate of interest is one that can be utilized by less than 5%(e.g., less than 3%, less than 2%, less than 1%, less than 0.5%, lessthan 0.2%, less than 0.1%, or none) of other bacterial cells present inthe organ. In some cases (e.g., in any of the methods of thedisclosure), the rare carbohydrate of interest is one that can beutilized by less than 2% (e.g., less than 1%, less than 0.5%, less than0.2%, less than 0.1%, or none) of other bacterial cells present in theorgan. In some cases (e.g., in any of the methods of the disclosure),the rare carbohydrate of interest is one that can be utilized by lessthan 0.5% (e.g., less than 0.2%, less than 0.1%, or none) of otherbacterial cells present in the organ. In some cases (e.g., in any of themethods of the disclosure), the rare carbohydrate of interest is onethat can be utilized by none of the other bacterial cells present in theorgan.

A method of the disclosure can include a step of administering to anindividual (e.g., a human) a carbohydrate of interest (e.g., a rarecarbohydrate of interest) that is utilized as a carbon source by asubject genetically modified bacterial cell present in an organ of theindividual (e.g., the gut), wherein less than 50% (e.g., less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, less than 3%,less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than0.1%, or none; e.g., less than 30%, less than 10%, less than 3%, lessthan 1%, less than 0.3%, less than 0.1%, less than 0.03%, less than0.01%, less than 0.003%, less than 0.001%, less than 0.0001%, or none)of other bacterial cells present in the organ utilize the rarecarbohydrate of interest as a carbon source. In some cases, such amethod includes a step of introducing the genetically modified bacterialcell into the organ of the individual, e.g., prior to administering thecarbohydrate of interest. In some embodiments, the organ (e.g., gut) ofthe individual includes at least 5 other bacterial species (other thanthe genetically modified bacteria), e.g., at least 8, at least 10, atleast 15, at least 20, at least 50, at least 100, at least 200, at least500, or at least 1,000 other bacterial species. In some cases, the organ(e.g., gut) of the individual includes at least 50 other bacterialspecies (other than the genetically modified bacteria). In some cases,the organ (e.g., gut) of the individual includes at least 100 otherbacterial species (other than the genetically modified bacteria).

In some cases, two or more privileged niches (e.g., in some cases usingdietary substrates from distinct geographic regions) can be used, e.g.,to increase the effectiveness of this strategy even further. Forexample, this may allow robust colonization in the event that one of thetwo rare substrates is utilized by an individual's endogenousmicrobiota.

ii. Colonization and Entrenchment

In some cases, growth modulation is used to allow a bacterial cell(e.g., population of bacterial cells) to colonize a target organ (e.g.,gut). Thus, provided are methods that facilitate colonization of abacterial cell (e.g., a population of bacterial cells) in an organ(e.g., gut) of an individual. By “colonize” it is meant that anintroduced bacterial cell (e.g., population of bacterial cells) canestablish a population of a desired abundance or level or can establisha large enough population in the target organ that the population isdetectable, despite the presence of already established bacterialpopulations. In some cases, the introduced bacteria reaches a ‘highlevel’ of colonization (i.e., a ‘high abundance’). In such cases, theintroduced bacteria reaches an abundance of 10⁵ CFU/μl or more (e.g.,10⁶ CFU/μl or more, 10⁷ CFU/μl or more) (e.g., by day 6 afterintroduction) (e.g., in the target organ). Thus, in some cases, anintroduced bacteria reaches an abundance of 10⁷ CFU/μl or more. In somecases, the introduced bacteria reaches a ‘low level’ of colonization(i.e., a ‘low abundance’), but the population is still detectable (e.g.,by day 6 after introduction). For example, in some cases, the introducedbacteria reaches an abundance in a range of from 10² CFU/μl to 10⁴CFU/μl (e.g., 10² CFU/μl, 10³ CFU/μl, 10⁴ CFU/μl) (e.g., by day 6 afterintroduction) (e.g., in the target organ).

In some embodiments, the introduced/genetically modified bacteriareaches an abundance such that it attains a population level of 10% ormore (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, or 90% or more) of total anaerobic CFUs(see e.g., FIG. 2) (e.g., by day 6 after introduction) (e.g., in thetarget organ). In some embodiments, the introduced bacteria reaches anabundance such the population attains a population level in a range offrom 10%-90% (e.g., from 10%-80%, 10%-70%, 10%-60%, 10%-50%, 20%-90%,20%-80%, 20%-70%, 20%-60%, 20%-50%, 30%-90%, 30%-80%, 30%-70%, 30%-60%,or 30%-50%) of total anaerobic CFUs (e.g., by day 6 after introduction)(e.g., in the target organ).

In some embodiments, the introduced/genetically modified bacteriareaches an abundance such that it attains a population level of 1% ormore (e.g., 2% or more, 5% or more, or 10% or more) of total bacterialcells in the organ (e.g., gut). In some embodiments, theintroduced/genetically modified bacteria reaches an abundance such thatit attains a population level in a range of from 1%-50% of totalbacterial cells in the organ (e.g., from 1%-40%, 1%-30%, 1%-20%, 1%-15%,2%-50%, 2%-40%, 2%-30%, 2%-20%, 2%-15%, 5%-50%, 5%-40%, 5%-30%, 5%-20%,or 5%-15% of total bacterial cells in the organ).

In some cases at least a portion of the population of bacterial cells inan organ (e.g., gut) is displaced as a result of modulating the growth(e.g., by administration of a carbohydrate of interest) of thegenetically modified bacterial cell. For example, in some cases 5% ormore (e.g., 10% or more, 15% or more, or 20% or more) of the populationof bacterial cells in an organ (e.g., gut) is displaced as a result ofmodulating the growth (e.g., by administration of a carbohydrate ofinterest) of the genetically modified bacterial cell. In some cases, thegenetically modified bacterial cell and the bacterial cells in thepopulation of bacterial cells are the same species. In some cases, thegenetically modified bacterial cell and the bacterial cells in thepopulation of bacterial cells are different species. In some cases, thegenetically modified bacterial cell is generated by providing abacterial cell from the population of bacterial cells and removing orinactivating one or more genes in the bacterial cell.

In some embodiments, colonization is stable for a long period of time.Thus, in some cases a genetically modified bacterial cell of thedisclosure becomes entrenched in the gut. The term “entrench” (e.g.,“entrenchment”) is used herein to refer to a situation in which anintroduced species becomes a stable/persistent member of the communityinto which it was introduced. For example, in some cases whenentrenchment is not accomplished, an introduced bacterial species (e.g.,introduced into the gut of an individual) might be cleared (reduced toundetectable levels) by approximately one week after introduction. Ifthere is not a niche available to the introduced bacteria, it mustcompete with species that are already established (entrenched), and thatshare the same niche with the introduced bacteria (i.e., the introducedbacteria would have to compete for resources with already establishedspecies).

In some cases, entrenchment is accomplished when an introduced bacteria(e.g., a wild type species not normally present in the target organ,e.g., gut; a genetically modified bacterial cell; and the like) remainsdetectable or at a desired abundance or population level in the organinto which it was introduced (e.g., on day 6 after introduction). Inother words, in some cases an introduced bacteria is entrenched if itremains detectable or at a desired abundance or population level for 6or more days (e.g., 7 or more, 8 or more, 9 or more, or 10 or more) daysafter introduction. By detectable it is meant that the population ofintroduced bacteria includes a large enough number of individuals thattheir presence is detectable. In some cases, entrenchment is maintainedby providing a sufficient amount of a carbohydrate (e.g., rarecarbohydrate of interest) that can be utilized by the geneticallymodified bacterial cell at a sufficient frequency. In some cases,entrenchment can be maintained in the absence of a carbohydrate (e.g.,rare carbohydrate of interest) that can be utilized by the geneticallymodified bacterial cell.

For any of the above metrics (e.g., abundance level, abundance range,population level, and the like), a time frame can be placed on themetric. For example, in some cases the metric is reached within 5 daysof introduction of the bacteria. In some case, the metric is reachedwithin 6 days of introduction of the bacteria. In some case, the metricis reached within 7 days of introduction of the bacteria. However, thetime frame need not be relative to introduction of the bacteria. Forexample, in some cases the metric is relative to the introduction of theresource (e.g., carbohydrate of interest, rare carbohydrate of interestsuch as porphyran). Thus, in some cases the metric is reached within 5days of introduction of the resource (e.g., carbohydrate of interest,rare carbohydrate of interest such as porphyran). In some case, somecases the metric is reached within 6 days of introduction of theresource (e.g., carbohydrate of interest, rare carbohydrate of interestsuch as porphyran). In some case, the metric is reached within 7 days ofintroduction of the resource (e.g., carbohydrate of interest, rarecarbohydrate of interest such as porphyran).

In some embodiments a method of the disclosure includes a step ofdetecting the presence of (e.g., measure the abundance of) thegenetically modified bacterial cell after it has been introduced into anindividual (e.g., 3 days, 4 days, 5 days, 6 days, and/or 7 days afterintroduction; and/or 3 days, 4 days, 5 days, 6 days, and/or 7 days afterproviding the carbohydrate of interest). In some such cases, the step ofdetecting includes measuring the colony forming units (e.g., CFU/μl) ofthe genetically modified bacterial cell present in the organ (e.g., gut)of the individual. In some such cases the detecting further includesmeasuring the colony forming units (e.g., CFU/μl) of other bacteriapresent in the organ.

In some cases, the term “controlled” is used in front of “colonization”and/or “entrenchment” (e.g., “controlled colonization”; “controlledentrenchment”). The term “controlled” is used as such to refer to asituation in which the colonization and/or entrenchment of a bacteria iscontrollable by a user. For example, if the growth and/or survival of agiven bacterial cell (e.g., a genetically modified bacterial cell) canbe modulated by administering an energy and/or carbon source (e.g.,carbohydrate of interest) to a host (e.g., a human) and/or a host organ,then the colonization and/or entrenchment of the bacterial iscontrollable. For example, in such a situation the population sizeand/or duration of colonization can depend on the amount and/orfrequency with which the energy and/or carbon source (e.g.,carbohydrate) is administered.

iii. Carbohydrate of Interest

In some embodiments the carbohydrate of interest (e.g., rarecarbohydrate of interest) is a polysaccharide. In some cases thecarbohydrate of interest (e.g., rare carbohydrate of interest) is asulfated carbohydrate. In some cases the carbohydrate of interest (e.g.,rare carbohydrate of interest) is selected from the group consisting ofporphyran, agarose, carrageenan, and any combination thereof. In somecases the carbohydrate of interest (e.g., rare carbohydrate of interest)is a carbohydrate cleaved by a glycoside hydrolase belonging toglycoside hydrolase family GH86. In some cases the carbohydrate ofinterest (e.g., rare carbohydrate of interest) is a marine carbohydrate.Examples of marine carbohydrates include but are not limited to:porphyran, agarose, agaropectin, carrageenan, and marine microbeexopolysaccharides. In some cases the carbohydrate of interest (e.g.,rare carbohydrate of interest) is selected from porphyran and agarose.In some cases the carbohydrate of interest (e.g., rare carbohydrate ofinterest) is porphyran. In some cases the carbohydrate of interest(e.g., rare carbohydrate of interest) is agarose.

In some cases the carbohydrate of interest (e.g., rare carbohydrate ofinterest) is a carbohydrate that contains a glycosidic linkage selectedfrom the group consisting of β-d-galactopyranose toα-l-galactopyranose-6-sulphate, β-d-galactopyranose to3,6-anhydro-α-l-galactopyranose.

In some cases the carbohydrate of interest (e.g., rare carbohydrate ofinterest) is a sulfated polygalactan. In some such cases, one or more ofthe galactose residues of the sulfated polygalactan can be a3,6-anhydro-galactose (e.g., in some cases joined by alternating α-1,3and β-1,4-glycosidic linkage). In some cases, one or more of thegalactopyranose residues of the sulfated polygalactan can be modified byone or more ester sulfates. In some cases, one or more of the galactoseresidues of the sulfated polygalactan is a 3,6-anhydro-galactose (e.g.,in some cases joined by alternating α-1,3 and β-1,4-glycosidic linkage);and one or more of the galactopyranose residues of the sulfatedpolygalactan is modified by one or more ester sulfates.

In some cases, the carbohydrate of interest is administered to anindividual and in some such cases the carbohydrate of interest isisolated. The term “isolated” refers to the state in which a molecule(e.g., a carbohydrate) can be. In such a case, the carbohydrate will befree or substantially free of material with which it is naturallyassociated such as other carbohydrates with which it is found in thenatural environment, or the environment in which it is prepared.

In some cases, less than 50% (e.g., less than 40%, less than 30%, lessthan 20%, less than 10%, less than 5%, less than 3%, less than 2%, lessthan 1%, less than 0.5%, less than 0.2%, less than 0.1%, or none; e.g.,less than 30%, less than 10%, less than 3%, less than 1%, less than0.3%, less than 0.1%, less than 0.03%, less than 0.01%, less than0.003%, less than 0.001%, less than 0.0001%, or none) of other bacterialcells present in the organ utilize the carbohydrate of interest (e.g.,rare carbohydrate of interest) as a carbon source. In some cases, thecarbohydrate of interest (e.g., rare carbohydrate of interest) cannot beutilized or digested by mammalian (e.g., human) cells.

In some cases, the carbohydrate of interest is administered at asufficient frequency and/or at a sufficient amount for controlledcolonization and/or controlled entrenchment. For example, thecarbohydrate of interest can be administered 1, 2, 3, 4, 5, 6, or moretimes per day, week, month, or year. The carbohydrate of interest can beadministered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredays, weeks, months, or years. In some cases, the carbohydrate ofinterest can be administered by oral administration. Any convenient typeof oral administration can be used. For example, oral administration caninclude delivery via eating (e.g., incorporated into food), drinking(e.g., incorporated into a solution such as drinking water), oral gavage(e.g., using a stomach tube), aerosol spray, tablets, capsules, pills,powders, and the like.

iv. Genetically Modified Bacteria

In some cases a genetically modified bacterial cell includes aheterologous carbohydrate-utilization gene or gene set. “Heterologous,”as used herein, means a nucleic acid and/or polypeptide that is notfound in the native bacteria. For example, in a case where a bacterialcell includes a gene set (e.g., a nucleic acid encoding a set ofproteins) that provides the cell with the ability to utilize a carbonsource that the cell otherwise cannot use, and where that gene set isnot naturally found in that bacteria, the gene set is teterologous' tothe bacterial cell.

In some cases a subject method is a method of genetically modifying abacterial cell to utilize as a carbon source a rare carbohydrate ofinterest. Any convenient method can be used to introduce a nucleic acidinto a prokaryotic cell, e.g., by electroporation (e.g., usingelectro-competent cells), by conjugation, by chemical methods (e.g.,using chemically competent cells), and the like.

In some embodiments, a genetically modified bacterial cell utilizes as acarbon source a rare carbohydrate of interest (as described elsewhereherein, e.g., wherein less than 50% of other bacterial cells present inthe organ utilize the rare carbohydrate of interest as a carbon source).This is due to the fact that a subject genetically modified bacterialcell includes a carbohydrate-utilization gene or gene set that providesthe genetically modified bacterial cell with the ability to use acarbohydrate of interest as a carbon source. In some cases, thecarbohydrate-utilization gene or gene set is native to the cell. In somecase, the carbohydrate-utilization gene or gene set is heterologous tothe cell (e.g., can be from a different species of bacteria). Thus, insome cases, the carbohydrate of interest cannot be utilized as a carbonsource by the genetically modified bacterial cell in the absence of aheterologous carbohydrate-utilization gene or gene set. In some cases,the genetically modified bacterial cell is capable of utilizing thecarbohydrate of interest as a carbon source in the absence of aheterologous carbohydrate-utilization gene or gene set, but the presenceof the heterologous carbohydrate-utilization gene or gene set enhancesthat capability.

In some cases, a carbohydrate-utilization gene or gene set includes aporphyranase (e.g., one from GH family 86 (GH86)). In some cases, acarbohydrate-utilization gene or gene set includes an agarase (e.g., onefrom GH family 86 (GH86)).

In some cases, a subject carbohydrate-utilization gene or gene setincludes one or more nucleic acids encoding BACPLE_1683-1706 from the B.plebeius genome (or homologs thereof) (see, e.g., Table 6). In somecases, a subject carbohydrate-utilization gene or gene set includes oneor more nucleic acids encoding BACPLE_1683-1687 from the B. plebeiusgenome (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding BACPLE_1700-1706 from the B. plebeius genome (or homologsthereof). In some cases, a subject carbohydrate-utilization gene or geneset includes one or more nucleic acids encoding BACPLE_1683-1687 fromthe B. plebeius genome (or homologs thereof) and BACPLE_1700-1706 fromthe B. plebeius genome (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding BACPLE_1683-1699 from the B. plebeius genome (or homologsthereof). In some cases, a subject carbohydrate-utilization gene or geneset includes one or more nucleic acids encoding BACPLE_1688-1706 fromthe B. plebeius genome (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding BACPLE_1683-1687 from the B. plebeius genome (or homologsthereof) as well as both porphyranases from within BACPLE_1688-1699 fromthe B. plebeius genome (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding BACPLE_1700-1706 from the B. plebeius genome (or homologsthereof) as well as both porphyranases from within BACPLE_1688-1699 fromthe B. plebeius genome (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding BACPLE_1683-1687 and BACPLE_1700-1706 from the B.plebeius genome (or homologs thereof) as well as both porphyranases fromwithin BACPLE_1688-1699 from the B. plebeius genome (or homologsthereof).

As such, in some cases, a subject carbohydrate-utilization gene or geneset includes one or more nucleic acids encoding proteins that have 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,or 100% sequence identity) with BACPLE_1683-1706 from the B. plebeiusgenome (see, e.g., Table 6). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1683-1687 from the B. plebeius genome. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1700-1706 from the B. plebeius genome. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1683-1687 from the B. plebeius genome (or homologs thereof) andBACPLE_1700-1706 from the B. plebeius genome. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1683-1699 from the B. plebeius genome. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1688-1706 from the B. plebeius genome. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withBACPLE_1683-1687 from the B. plebeius genome (or homologs thereof) aswell as both porphyranases from within BACPLE_1688-1699 from the B.plebeius genome. In some cases, a subject carbohydrate-utilization geneor gene set includes one or more nucleic acids encoding proteins thathave 80% or more sequence identity (e.g., 85% or more, 90% or more, 95%or more, or 100% sequence identity) with BACPLE_1700-1706 from the B.plebeius genome (or homologs thereof) as well as both porphyranases fromwithin BACPLE_1688-1699 from the B. plebeius genome. In some cases, asubject carbohydrate-utilization gene or gene set includes one or morenucleic acids encoding proteins that have 80% or more sequence identity(e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity)with BACPLE_1683-1687 and BACPLE_1700-1706 from the B. plebeius genome(or homologs thereof) as well as both porphyranases from withinBACPLE_1688-1699 from the B. plebeius genome.

TABLE 6 SEQ ID NOs. and annotations for proteins encoded by B. plebiusgenome (BACPLE_1669-1706). B. Plebeius Homolog ID SEQ ID NO AnnotationBACPLE_01669 1 histidine kinase CDS BACPLE_01670 2 beta-agarase CDSBACPLE_01671 3 glycosyhydrolase CDS BACPLE_01672 4 threonine synthaseCDS BACPLE_01673 5 hypothetical protein CDS BACPLE_01674 6 altronatehydrolase CDS BACPLE_01675 7 altronate oxidoreductase CDS BACPLE_01676 8sorbitol dehydrogenase CDS BACPLE_01677 9 L-fucose: H+ symporterpermease CDS BACPLE_01678 10 amidohydrolase CDS BACPLE_01679 11aldo/keto reductase CDS BACPLE_01680 12 hypothetical protein CDSBACPLE_01682 13 hypothetical protein CDS BACPLE_01683 14 hypotheticalprotein CDS BACPLE_01684 15 glycosyhydrolase CDS BACPLE_01685 16hypothetical protein CDS BACPLE_01686 17 hypothetical protein CDSBACPLE_01688 18 hypothetical protein CDS BACPLE_01689 19beta-porphyranase B CDS BACPLE_01692 20 hypothetical protein CDSBACPLE_01693 21 beta-porphyranase A CDS BACPLE_01694 22 hypotheticalprotein CDS BACPLE_01695 23 hypothetical protein CDS BACPLE_01696 24hypothetical protein CDS BACPLE_01697 25 hypothetical protein CDSBACPLE_01698 26 SusC/RagA family TonB-linked outer membrane protein CDSBACPLE_01699 27 hybrid two component system BACPLE_01700 28 alcoholdehydrogenase CDS BACPLE_01701 29 acetylglucosamine-6-sulfatase CDSBACPLE_01702 30 hypothetical protein CDS BACPLE_01703 31 glycosidehydrolase CDS BACPLE_01704 32 hypothetical protein CDS BACPLE_01705 33hypothetical protein CDS BACPLE_01706 34 beta-galactosidase CDS

In some cases, a subject carbohydrate-utilization gene or gene setincludes one or more nucleic acids encoding SEQ ID NOs.: 14-34 (orhomologs thereof) (see, e.g., Table 6). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding SEQ ID NOS.: 14-17 (or homologs thereof). In some cases,a subject carbohydrate-utilization gene or gene set includes one or morenucleic acids encoding SEQ ID NOS.: 28-34 (or homologs thereof). In somecases, a subject carbohydrate-utilization gene or gene set includes oneor more nucleic acids encoding SEQ ID NOS.: 14-17 (or homologs thereof)and SEQ ID NOS.: 28-34 (or homologs thereof). In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding SEQ ID NOS.: 14-27 (or homologs thereof). In some cases,a subject carbohydrate-utilization gene or gene set includes one or morenucleic acids encoding SEQ ID NOS.: 18-34 (or homologs thereof). In somecases, a subject carbohydrate-utilization gene or gene set includes oneor more nucleic acids encoding SEQ ID NOS.: 14-17 (or homologs thereof)as well as both porphyranases from within SEQ ID NOS.: 18-27 (orhomologs thereof). In some cases, a subject carbohydrate-utilizationgene or gene set includes one or more nucleic acids encoding SEQ IDNOS.: 28-34 (or homologs thereof) as well as both porphyranases fromwithin SEQ ID NOS.: 18-27 (or homologs thereof). In some cases, asubject carbohydrate-utilization gene or gene set includes one or morenucleic acids encoding SEQ ID NOS.: 14-17 and SEQ ID NOS.: 28-34 (orhomologs thereof) as well as both porphyranases from within SEQ ID NOS.:18-27 (or homologs thereof).

As such, in some cases, a subject carbohydrate-utilization gene or geneset includes one or more nucleic acids encoding proteins that have 80%or more sequence identity (e.g., 85% or more, 90% or more, 95% or more,or 100% sequence identity) with SEQ ID NOS.: 14-34 (see, e.g., Table 6).In some cases, a subject carbohydrate-utilization gene or gene setincludes one or more nucleic acids encoding proteins that have 80% ormore sequence identity (e.g., 85% or more, 90% or more, 95% or more, or100% sequence identity) with SEQ ID NOS.: 14-17. In some cases, asubject carbohydrate-utilization gene or gene set includes one or morenucleic acids encoding proteins that have 80% or more sequence identity(e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity)with SEQ ID NOS.: 28-34. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withSEQ ID NOS.: 14-17 and SEQ ID NOS.: 28-34. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withSEQ ID NOS.: 14-27. In some cases, a subject carbohydrate-utilizationgene or gene set includes one or more nucleic acids encoding proteinsthat have 80% or more sequence identity (e.g., 85% or more, 90% or more,95% or more, or 100% sequence identity) with SEQ ID NOS.: 18-34. In somecases, a subject carbohydrate-utilization gene or gene set includes oneor more nucleic acids encoding proteins that have 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequenceidentity) with SEQ ID NOS.: 14-17 as well as both porphyranases setforth as SEQ ID NOs.: 19 and 21. In some cases, a subjectcarbohydrate-utilization gene or gene set includes one or more nucleicacids encoding proteins that have 80% or more sequence identity (e.g.,85% or more, 90% or more, 95% or more, or 100% sequence identity) withSEQ ID NOS.: 28-34 as well as both porphyranases set forth as SEQ IDNOs.: 19 and 21. In some cases, a subject carbohydrate-utilization geneor gene set includes one or more nucleic acids encoding proteins thathave 80% or more sequence identity (e.g., 85% or more, 90% or more, 95%or more, or 100% sequence identity) with SEQ ID NOS.: 14-17 and SEQ IDNOS.: 28-34 as well as both porphyranases set forth as SEQ ID NOs.: 19and 21.

In some cases, a subject carbohydrate-utilization gene or gene setincludes one or more nucleic acids encoding (i) at least one of SEQ IDNOs.: 19, 21, and 22 (or a homolog(s) thereof); (ii) at least one of SEQID NOs.: 26 and 33 (or a homolog(s) thereof); and (iii) at least one ofSEQ ID NOs.: 25 and 32 (or a homolog(s) thereof). In some cases, asubject carbohydrate-utilization gene or gene set encodes SEQ ID NOs.:19, 21-22, 25, 26, and 32-33 (or a homologs thereof).

In some cases, a subject carbohydrate-utilization gene or gene setincludes one or more nucleic acids encoding (i) at least one proteinthat has 80% or more sequence identity (e.g., 85% or more, 90% or more,95% or more, or 100% sequence identity) with any one of SEQ ID NOs.: 19,21, and 22; (ii) at least one protein that has 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequenceidentity) with any one of SEQ ID NOs.: 26 and 33; and (iii) at least oneprotein that has 80% or more sequence identity (e.g., 85% or more, 90%or more, 95% or more, or 100% sequence identity) with any one of SEQ IDNOs.: 25 and 32. In some cases, a subject carbohydrate-utilization geneor gene set includes one or more nucleic acids encoding proteins having80% or more sequence identity (e.g., 85% or more, 90% or more, 95% ormore, or 100% sequence identity) with SEQ ID NOs.: 19, 21-22, 25, 26,and 32-33.

In some embodiments, the carbohydrate-utilization gene set includes atleast 3 genes (e.g., at least 4, at least 5, at least 6, at least 8genes, at least 10 genes, at least 12 genes, at least 15 genes, or atleast 20 genes). In some cases, the carbohydrate-utilization gene setincludes from 3-30 genes (e.g., from 5-30, 3-25, 3-20, 3-15, 3-10, 3-8,5-25, 5-20, 5-15, 5-10, 5-8, 8-30, 8-25, 8-20, 8-15, 10-30, 10-25,10-20, 10-15, 12-30, 12-25, 12-20, 15-30, 15-25, 20-30, or 20-25 genes).In some cases, the carbohydrate-utilization gene set includes from 3-10genes.

A nucleic acid that includes the carbohydrate-utilization gene or geneset (e.g., a heterologous carbohydrate-utilization gene or gene set) mayor may not be integrated (covalently linked) into the genome of thecell. For example, in some cases, the nucleic acid that includes thecarbohydrate-utilization gene or gene set is integrated into the genomeof the cell (as a chromosomal integrant). In some cases, the nucleicacid that includes the carbohydrate-utilization gene or gene set ismaintained on an episomal element (extra chromosomal element) such as aplasmid.

In some cases the expression of genes from the carbohydrate-utilizationgene or gene set is controlled by a high expression promoter. In somecases the expression of genes from the carbohydrate-utilization gene orgene set is controlled by an inducible promoter, constitutive promoter,native promoter (e.g., native to the bacterial cell), heterologouspromoter, or a promoter associated with the carbohydrate-utilizationgene or gene set. Thus, for example, methods of the disclosure can becontrolled by the type of promoter that is present. As an illustrativeexample, in some cases, entrenchment (e.g., of a subject geneticallymodified bacterial cell into an organ of an individual, e.g., into theindividual's gut) is controlled by a high expression promoter. Likewise,in some cases entrenchment (e.g., of a subject genetically modifiedbacterial cell into an organ of an individual, e.g., into theindividual's gut) is controlled by an inducible promoter, constitutivepromoter, native promoter (e.g., native to the bacterial cell),heterologous promoter, or a promoter associated with thecarbohydrate-utilization gene or gene set.

Therapy etc.

The strategy disclosed here for facilitating robust colonization canfacilitate a range of therapeutic applications, e.g., where it can beimportant to establish a high and/or predictable number of natural orengineered therapeutic microbes. For example, it may be important tocolonize an organ (e.g., gut) at a high enough level to ensure thattherapeutic activities may overcome competing activities of othermicrobes. As an example, this may be important when attempting to, forinstance, change the short chain fatty acid profile or reduce theaccumulation of harmful chemicals that are produced in the gut.Achieving predictable colonization can also be important fortherapeutics that must be carefully dosed, such as bacteria with pro- oranti-inflammatory activities which could be used as an adjuvant forcancer immunotherapy or in the treatment of Inflammatory Bowel Diseases.The methods of the disclosure could also be used to, for example, ensurerobust colonization to displace a harmful species by establishing anon-harmful version that can effectively compete for limiting resourceswith the harmful strain. As such, and as noted elsewhere, in some casesa subject method can result in displacement of bacteria present in thegut of an individual.

A subject genetically modified bacterial cell can be introduced into avariety of individuals with a variety of ailments. Diseases that can betreated with a subject genetically modified bacterial cell include butare not limited to diseases that are impacted by the gut microbiota,which include obesity, diabetes, heart disease, central nervous systemdiseases, rheumatoid arthritis, metabolic disorders, and cancer. Forexample, in some cases, the individual has gut inflammation, and in somesuch cases the individual has an inflammatory disease (e.g., Crohn'sdisease, ulcerative colitis, and the like), and in some cases gutinflammation can indirectly impact the disease, such as colorectalcancer or obesity.

In some cases, a subject bacterial cell is a therapeutic cell (has apositive impact on a clinical parameter of the individual). Atherapeutic cell is not necessarily a genetically modified cell. Forexample, a cell that has not been genetically modified may serve atherapeutic purpose to an individual. In some cases, a therapeutic cellis genetically modified. For example, in some cases a therapeutic cellincludes a transgene that encodes a therapeutic peptide (e.g., a peptidethat when expressed by—and in some cases secreted by—a cell can have apositive impact on the health of an individual). For example, atherapeutic cell and/or therapeutic peptide can have antimicrobial(antibiotic) activity (e.g., against one or more gut microbes), canfunction to change gut environmental parameters (e.g., pH control), canaffect inflammation, can provide an enzymatic activity, and the like.

In some cases, a genetically modified bacterial cell includes atransgene that is an enzyme (e.g., a metabolic enzyme). For example,there are many small molecules produced in the gut (e.g., produced bymicrobes) that accumulate in the blood and cause or exacerbate diseases.Expressing an enzyme or a pathway (as a transgene) in a Bacteroides cell(or population of cells) to break down these products can be used inmethods of treatment. For example, a Bacteroides cell expressing such atransgene can be introduced into the gut of an individual (e.g., inorder to break down small molecules, which in some cases may be producedby microbes, to reduce or even eliminate the amount absorbed by the gutof the individual, reducing the accumulation of the molecules in theblood of the individual).

In some cases, a genetically modified bacterial cell is tagged (e.g., toaid in tracking). For example, in some cases, a genetically modifiedbacterial cell of the disclosure includes a detectable label (e.g., anucleic acid that results in the presence of a detectable signal). Assuch, in some cases, a subject genetically modified cell (or populationof cells) that is introduced into an individual (e.g., the gut),includes a transgene whose expression detectably labels the cell. Thephrase “detectably label” as used herein refers to any expressionproduct (RNA, protein) that is detectable. The expression product (thelabel) can itself be detectable (directly detectable label) (e.g., afluorescent protein), or the label can be indirectly detectable, e.g.,in the case of an enzymatic label, the enzyme (e.g., luciferase orrecombinase) may catalyze a chemical alteration of a substrate compound,composition or nucleic acid sequence and the product of the reaction isdetectable (e.g. by fluorescence, chemiluminescence, sequencing orpresence and/or size of a PCR product). Thus in some cases, any of themethods of the disclosure can include a step of detecting (e.g.,measuring) a detectable signal produced by a subject geneticallymodified bacterial cell. As an illustrative example, in some cases agenetically modified bacterial cell includes a transgene that encodes afluorescent protein (e.g., green fluorescent protein (GFP) or any of anumber of derivatives of GFP such as YFP, CFP, RFP, etc.) or an enzymethat produces a fluorescent signal (e.g., luciferase), and a subjectmethod includes a step of measuring the signal to detect the presence of(e.g., measure the abundance of) the genetically modified bacterial cellafter it has been introduced into an individual (e.g., 3 days, 4 days, 5days, 6 days, and/or 7 days after introduction; and/or 3 days, 4 days, 5days, 6 days, and/or 7 days after providing the carbohydrate ofinterest).

In some cases, a genetically modified bacterial cell includes atransgene that is a “marker” or “marker gene” or “marker protein.” Amarker is an expression product (e.g., mRNA, protein, non-coding RNA)that marks a host cell such that the host cell is detectable (e.g.,detectably labeled). In some cases, the host cell is detectable byvirtue of survival (e.g., the marker can be a “selectable marker”). Insome cases, the host cell is detectable by observation (e.g., by directvisualization, by performing an assay, by performing a measurement step,and the like) and the marker can be referred to as a “reporter” or“reporter gene” or “reporter protein.”

As noted above, some markers are “selectable markers.” Selectablemarkers (a “selectable marker gene” can encode a “selectable markerprotein”) provide for selection, i.e., for selective retention of cells(e.g., prokaryotic cells) that comprise the selectable marker gene,during culturing and propagation of the cells. An example of aselectable marker is a transgene that encodes a drug selectable markerprotein that provides drug resistance for prokaryotic cells (e.g.,Bacteroides cells). Such a selectable marker encodes a drug selectablemarker protein that provides resistance for prokaryotic cells to one ormore drugs (e.g., kanamycin, neomycin, ampicillin, carbenicillin,chloramphenicol, gentamicin, tetracycline, rifampin, trimethoprim,hygromycin B, spectinomycin, and the like). In some cases, a subjectgenetically modified bacteria can include an erythromycin resistancecassette. Proteins that provide drug resistance to cells (e.g.,prokaryotic cells) in which they are expressed are known in the art. Forexample, wild type genes/proteins are known that provide resistance(e.g., for prokaryotic cells) to the above drugs. For example,aminoglycoside 3′-phosphotransferase (APH), is a wild type protein thatprovides for resistance to the drugs Kanamycin, Neomycin and Geneticin(G418); while beta-lactamase is a wild type protein that provides forresistance to the drugs ampicillin and carbenecillin. Chloramphenicolacetyltransferase (cat) confers resistance to chloramphenicol. Genesconferring resistance to aminoglycosides include aac, aad, aph andstrA/B. Genes conferring resistance to β-lactams include ampC, cmy, temand vim. Genes conferring resistance to sulfonamides include sulI andsulII. Genes conferring resistance to tetracycline include tet(A),tet(B), tet(C), tet(D) and regulator, and tetR. Selectable markers canalso be those useful in balanced lethal systems, e.g., in which anessential gene is maintained on a plasmid with a correspondingchromosomal deletion or suppressible mutation on the host cell genome,e.g. a tRNA selectable marker that suppresses a host chromosomal genemutation; those useful in repressor titration systems, in which anoperator sequences, e.g. the lac operator or tet operator, placed on aplasmid, derepresses a chromosomal gene; antidote/poison selectionschemes, in which an antidote to a poison expressed from the hostchromosome (e.g. the ccdB gene) is maintained on the plasmid; and thoseuseful in RNA-based selection schemes, e.g. antisense regulators, orantisense regulators that inhibit the translation of a gene transcribedfrom the host chromosome that would otherwise promote cell death.

Bacteroides Cells

In any of the embodiments of the disclosure, the bacterial cell ofinterest (e.g., the genetically modified cell, the cell being modifiedor introduced, or whose growth is being modulated, as part of a subjectcomposition or method) can be a Bacteroides cell. In some cases, thebacterial cell of interest is not a Bacteroides cell. For example, thebacterial cell of interest can be any desired species, e.g., when thetarget organ is a gut the bacterial cell can be any species that cancolonize a gut. In some cases the bacterial cell of interest is aClostridium species (i.e., is a cell of the genus Clostridium).

The term “Bacteroides cell” is used herein to refer to a cell of thegenus Bacteroides. As such, in some cases, a subject cell is aBacteroides cell. Examples of species within the genus Bacteroidesinclude but are not limited to: B. fragilis (Bf), B. distasonis (Bd), B.thetaiotaomicron (Bt), B. vulgatus (By), B. ovatus (Bo), B. eggerrthii(Be), B. merdae (Bm), B. stercoris (Bs), B. uniformis (Bu), and B.caccae (Bc). In some cases, a Bacteroides cell is a species selectedfrom: B. acidifaciens, B. amylophilus, B. asaccharolyticus, B. bamesiae,B. bivius, B. buccae, B. buccalis, B. caccae, B. capillosus, B.capillus, B. cellulosilyticus, B. cellulosolvens, B. chinchilla, B.clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B.corporis, B. denticola, B. disiens, B. distasonis, B. dorei, B.eggerthii, B. endodontalis, B. faecichinchillae, B. faecis, B.finegoldii, B. fluxus, B. forsythus, B. fragilis, B. furcosus, B.galacturonicus, B. gallinarum, B. gingivalis, B. goldsteinii, B.gracilis, B. graminisolvens, B. helcogenes, B. heparinolyticus, B.hypermegas, B. intermedius, B. intestinalis, B. johnsonii, B. levvi, B.loescheii, B. macacae, B. massiliensis, B. melaninogenicus, B. merdae,B. microfusus, B. multiacidus, B. nodosus, B. nordii, B. ochraceus, B.oleiciplenus, B. oralis, B. oris, B. oulorum, B. ovatus, B.paurosaccharolyticus, B. pectinophilus, B. pentosaceus, B. plebeius, B.pneumosintes, B. polypragmatus, B. praeacutus, B. propionicifaciens, B.putredinis, B. pyogenes, B. reticulotermitis, B. rodentium, B.ruminicola, B. salanitronis, B. salivosus, B. salyersiae, B. sartorii,B. splanchnicus, B. stercorirosoris, B. stercoris, B. succinogenes, B.suis, B. tectus, B. termitidis, B. thetaiotaomicron, B. uniformis, B.ureolyticus, B. veroralis, B. vulgatus, B. xylanisolvens, B.xylanolyticus, and B. zoogleoformans. In some cases, a subjectBacteroides cell is a species selected from: B. fragilis (Bf), B.thetaiotaomicron (Bt), B. vulgatus (By), B. ovatus (Bo), and B.uniformis (Bu).

In some cases, the Bacteroides cell is a species selected from: B.fragilis (Bf), B. distasonis (Bd), B. thetaiotaomicron (Bt), B. vulgatus(By), B. ovatus (Bo), B. eggerrthii (Be), B. merdae (Bm), B. stercoris(Bs), B. uniformis (Bu), and B. caccae (Bc). In some cases, theBacteroides cell is a species selected from: B. fragilis (Bf), B.thetaiotaomicron (Bt), B. vulgatus (By), B. ovatus (Bo), and B.uniformis (Bu). In some cases, the Bacteroides cell is a speciesselected from: B. thetaiotaomicron (Bt), B. vulgatus (By), B. ovatus(Bo), and B. uniformis (Bu). In some cases, the Bacteroides cell is B.thetaiotaomicron (Bt).

Kits

Also provided are kits, e.g., for practicing any of the above methods.The contents of the subject kits may vary greatly. A kit can include,for example: (i) a subject genetically modified bacterial cell, and (ii)a rare carbohydrate of interest that can be utilized by the geneticallymodified bacterial cell. In some case, the genetically modifiedbacterial cell is a therapeutic cell.

In addition to the above components, the subject kits can furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, flash drive, etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the internet to access the information at aremoved site. Any convenient means may be present in the kits.

EXAMPLES OF NON-LIMITING ASPECTS OF THE DISCLOSURE

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-98 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

1. A method, comprising: introducing to a human organ a geneticallymodified bacterial cell capable of controlled entrenchment and/orcontrolled colonization.2. A method for modulating growth of a genetically modified bacterialcell present in a human organ, comprising: administering to a human arare carbohydrate of interest that is utilized as a carbon source by thegenetically modified bacterial cell, wherein less than 50% (e.g., lessthan 30%, less than 10%, less than 3%, less than 1%, less than 0.3%,less than 0.1%, less than 0.03%, less than 0.01%, less than 0.003%, lessthan 0.001%, less than 0.0001%, or none) of other bacterial cellspresent in the human organ utilize the rare carbohydrate of interest asa carbon source.3. A method for modulating growth of a genetically modified bacterialcell in an organ, comprising: introducing the genetically modifiedbacterial cell to the organ in vivo, wherein the genetically modifiedbacterial cell utilizes as a carbon source a rare carbohydrate ofinterest, wherein less than 50% (e.g., less than 30%, less than 10%,less than 3%, less than 1%, less than 0.3%, less than 0.1%, less than0.03%, less than 0.01%, less than 0.003%, less than 0.001%, less than0.0001%, or none) of other bacterial cells present in the organ utilizethe rare carbohydrate of interest as a carbon source.4. A method for displacing at least a portion of a population ofbacterial cells in an organ, comprising: introducing to the organ agenetically modified bacterial cell capable of controlled entrenchmentand/or controlled colonization, and displacing at least a portion of thepopulation of bacterial cells with the genetically modified bacterialcell.5. The method of 4, wherein the genetically modified bacterial cell andthe bacterial cells in the population of bacterial cells are the samespecies.6. The method of 4 or 5, further comprising providing a bacterial cellfrom the population of bacterial cells and removing or inactivating oneor more genes in the bacterial cell, thereby generating the geneticallymodified bacterial cell.7. The method of 3 or 4, wherein the organ is a human organ.8. The method of any one of the preceding, wherein the organ is a gut.9. The method of 1 or 4, wherein the controlled entrenchment and/orcontrolled colonization is controlled by a high expression promoter.10. The method of 1 or 4, wherein the controlled entrenchment and/orcontrolled colonization is controlled by an inducible promoter.11. The method of 1 or 4, wherein the controlled entrenchment and/orcontrolled colonization is controlled by delivering a rare carbohydrateof interest to the organ.12. The method of 11, wherein the rare carbohydrate of interest is acarbohydrate that can be utilized as a carbon source by less than 50%(e.g., less than 30%, less than 10%, less than 3%, less than 1%, lessthan 0.3%, less than 0.1%, less than 0.03%, less than 0.01%, less than0.003%, less than 0.001%, less than 0.0001%, or none) of other bacterialcells present in the organ, thereby generating a privileged niche.13. The method of any one of 2-3 and 11-12, wherein the rarecarbohydrate of interest is an isolated carbohydrate.14. The method of any one of 2-3 and 11-13, wherein the rarecarbohydrate of interest is a polysaccharide.15. The method of any one of 2-3 and 11-14, wherein the rarecarbohydrate of interest is a sulfated carbohydrate.16. The method of any one of 2-3 and 11-14, wherein the rarecarbohydrate of interest is selected from the group consisting ofporphyran, agarose, carrageenan, and any combination thereof.17. The method of any one of 2-3 and 11-16, wherein the rarecarbohydrate of interest is a carbohydrate cleaved by a glycosidehydrolase belonging to glycoside hydrolase family GH86.18. The method of any one of 2-3 and 11-17, wherein the rarecarbohydrate of interest is a sulfated polygalactan.19. The method of any one of the preceding, wherein the geneticallymodified bacterial cell is a genetically modified gut resident bacterialcell.20. The method of any one of the preceding, wherein the geneticallymodified bacterial cell is in the genus Bacteroides.21. The method of any one of the preceding, wherein the geneticallymodified bacterial cell is selected from B. acidifaciens, B.amylophilus, B. asaccharolyticus, B. barnesiae, B. bivius, B. buccae, B.buccalis, B. caccae, B. capillosus, B. capillus, B. cellulosilyticus, B.cellulosolvens, B. chinchilla, B. clarus, B. coagulans, B. coprocola, B.coprophilus, B. coprosuis, B. corporis, B. denticola, B. disiens, B.distasonis, B. dorei, B. eggerthii, B. endodontalis, B.faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. forsythus, B.fragilis, B. furcosus, B. galacturonicus, B. gallinarum, B. gingivalis,B. goldsteinii, B. gracilis, B. graminisolvens, B. helcogenes, B.heparinolyticus, B. hypermegas, B. intermedius, B. intestinalis, B.johnsonii, B. levvi, B. loescheii, B. macacae, B. massiliensis, B.melaninogenicus, B. merdae, B. microfusus, B. multiacidus, B. nodosus,B. nordii, B. ochraceus, B. oleiciplenus, B. oralis, B. oris, B.oulorum, B. ovatus, B. paurosaccharolyticus, B. pectinophilus, B.pentosaceus, B. plebeius, B. pneumosintes, B. polypragmatus, B.praeacutus, B. propionicifaciens, B. putredinis, B. pyogenes, B.reticulotermitis, B. rodentium, B. ruminicola, B. salanitronis, B.salivosus, B. salyersiae, B. sartorii, B. splanchnicus, B.stercorirosoris, B. stercoris, B. succinogenes, B. suis, B. tectus, B.termitidis, B. thetaiotaomicron, B. uniformis, B. ureolyticus, B.veroralis, B. vulgatus, B. xylanisolvens, B. xylanolyticus, B.zoogleoformans, and any combination thereof.22. The method of any one of the preceding, wherein the geneticallymodified bacterial cell comprises a carbohydrate-utilization gene orgene set.23. The method of 22, wherein the carbohydrate-utilization gene or geneset comprises one or more genes selected from the group consisting ofporphyranase, glycoside hydrolase, sulfatase, galactosidase, and anycombination thereof.24. The method of 22, wherein the carbohydrate-utilization gene or geneset comprises one or more nucleic acids encoding proteins that have 80%or more sequence identity with BACPLE_1683-1706 from the B. plebeiusgenome.25. The method of any one of 22-24, wherein the carbohydrate-utilizationgene set comprises at least six genes.26. The method of any one of 22-25, wherein the carbohydrate of interestcannot be utilized as a carbon source by the genetically modifiedbacterial cell in the absence of the carbohydrate-utilization gene orgene set.27. The method of any one of 22-26, wherein the carbohydrate-utilizationgene or gene set is heterologous.28. The method of 27, wherein the carbohydrate-utilization gene or geneset is encoded on a plasmid, encoded on a bacterial artificialchromosome, or genomically integrated.29. The method of any one of the preceding, wherein the geneticallymodified bacterial cell is capable of treating a metabolic disease ordisorder.30. The method of any one of the preceding, wherein the geneticallymodified bacterial cell comprises one or more therapeutic transgenes.31. The method of 30, wherein the one or more therapeutic transgenescomprise one or more enzymes.32. The method of any one of 2-3 and 11-18, further comprisingadministering the rare carbohydrate of interest for a sufficient amountof time and at a sufficient frequency to establish a population of thegenetically modified bacterial cell.33. The method of 32, further comprising maintaining the population ofthe genetically modified bacterial cell in the organ for at least 5days.34. The method of any one of 2-3 and 11-18, further comprisingadministering the rare carbohydrate of interest for at least 3 days.35. The method of any one of the preceding, wherein the geneticallymodified bacterial cell is introduced orally.36. The method of any one of the preceding, wherein the organ comprisesat least 5 other bacterial species.37. The method of any one of the preceding, further comprisingcolonizing the organ with the genetically modified bacterial cell at alevel of at least 1% of total bacterial cells in the organ.38. A genetically modified bacterial cell that utilizes as a carbonsource a rare carbohydrate of interest that is utilized as a carbonsource by less than 50% (e.g., less than 30%, less than 10%, less than3%, less than 1%, less than 0.3%, less than 0.1%, less than 0.03%, lessthan 0.01%, less than 0.003%, less than 0.001%, less than 0.0001%, ornone) of bacterial cells present in a human microbiota.39. A genetically modified bacterial cell, comprising:

a heterologous carbohydrate-utilization gene or gene set that providesthe genetically modified bacterial cell with an ability to utilize as acarbon source a rare carbohydrate of interest that is utilized as acarbon source by less than 50% (e.g., less than 30%, less than 10%, lessthan 3%, less than 1%, less than 0.3%, less than 0.1%, less than 0.03%,less than 0.01%, less than 0.003%, less than 0.001%, less than 0.0001%,or none) of bacterial cells present in a human microbiota.

40. A genetically modified bacterial cell, comprising:(i) a heterologous therapeutic transgene; and(ii) a carbohydrate-utilization gene or gene set that provides thegenetically modified bacterial cell with an ability to utilize as acarbon source a rare carbohydrate of interest that is utilized as acarbon source by less than 50% (e.g., less than 30%, less than 10%, lessthan 3%, less than 1%, less than 0.3%, less than 0.1%, less than 0.03%,less than 0.01%, less than 0.003%, less than 0.001%, less than 0.0001%,or none) of bacterial cells present in a human microbiota.41. The genetically modified bacterial cell of any one of 38-40, whereinthe rare carbohydrate of interest is an isolated carbohydrate.42. The genetically modified bacterial cell of any one of 38-41, whereinthe rare carbohydrate of interest is a polysaccharide.43. The genetically modified bacterial cell of any one of 38-42, whereinthe rare carbohydrate of interest is a sulfated carbohydrate.44. The genetically modified bacterial cell of any one of 38-42, whereinthe rare carbohydrate of interest is selected from the group consistingof porphyran, agarose, carrageenan, and any combination thereof.45. The genetically modified bacterial cell of any one of 38-44, whereinthe rare carbohydrate of interest is a carbohydrate cleaved by aglycoside hydrolase belonging to glycoside hydrolase family GH86.46. The genetically modified bacterial cell of any one of 38-45, whereinthe rare carbohydrate of interest is a sulfated polygalactan.47. The genetically modified bacterial cell of any one of 38-46, whereinthe genetically modified bacterial cell is a genetically modified gutresident bacterial cell.48. The genetically modified bacterial cell of any one of 38-47, whereinthe genetically modified bacterial cell is in the genus Bacteroides.49. The genetically modified bacterial cell of any one of 38-48, whereinthe genetically modified bacterial cell is selected from B.acidifaciens, B. amylophilus, B. asaccharolyticus, B. barnesiae, B.bivius, B. buccae, B. buccalis, B. caccae, B. capillosus, B. capillus,B. cellulosilyticus, B. cellulosolvens, B. chinchilla, B. clarus, B.coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. corporis, B.denticola, B. disiens, B. distasonis, B. dorei, B. eggerthii, B.endodontalis, B. faecichinchillae, B. faecis, B. finegoldii, B. fluxus,B. forsythus, B. fragilis, B. furcosus, B. galacturonicus, B.gallinarum, B. gingivalis, B. goldsteinii, B. gracilis, B.graminisolvens, B. helcogenes, B. heparinolyticus, B. hypermegas, B.intermedius, B. intestinalis, B. johnsonii, B. lewi, B. loescheii, B.macacae, B. massiliensis, B. melaninogenicus, B. merdae, B. microfusus,B. multiacidus, B. nodosus, B. nordii, B. ochraceus, B. oleiciplenus, B.oralis, B. oris, B. oulorum, B. ovatus, B. paurosaccharolyticus, B.pectinophilus, B. pentosaceus, B. plebeius, B. pneumosintes, B.polypragmatus, B. praeacutus, B. propionicifaciens, B. putredinis, B.pyogenes, B. reticulotermitis, B. rodentium, B. ruminicola, B.salanitronis, B. salivosus, B. salyersiae, B. sartorii, B. splanchnicus,B. stercorirosoris, B. stercoris, B. succinogenes, B. suis, B. tectus,B. termitidis, B. thetaiotaomicron, B. uniformis, B. ureolyticus, B.veroralis, B. vulgatus, B. xylanisolvens, B. xylanolyticus, B.zoogleoformans, and any combination thereof.50. The genetically modified bacterial cell of 38, wherein thegenetically modified bacterial cell comprises a carbohydrate-utilizationgene or gene set.51. The genetically modified bacterial cell of 39, 40, or 50, whereinthe carbohydrate-utilization gene or gene set comprises one or moregenes selected from the group consisting of porphyranase, glycosidehydrolase, sulfatase, galactosidase, and any combination thereof.52. The genetically modified bacterial cell of any one of 39-40 and50-51, wherein the carbohydrate-utilization gene or gene set comprisesone or more nucleic acids encoding proteins that have 80% or moresequence identity with BACPLE_1683-1706 from the B. plebeius genome.53. The genetically modified bacterial cell of any one of 39-40 and50-52, wherein the carbohydrate-utilization gene set comprises at leastsix genes.54. The genetically modified bacterial cell of any one of 39-40 and50-53, wherein the carbohydrate of interest cannot be utilized as acarbon source by the genetically modified bacterial cell in the absenceof the carbohydrate-utilization gene or gene set.55. The genetically modified bacterial cell of 40 or 50, wherein thecarbohydrate-utilization gene or gene set is heterologous.56. The genetically modified bacterial cell of 39 or 55, wherein thecarbohydrate-utilization gene or gene set is encoded on a plasmid,encoded on a bacterial artificial chromosome, or genomically integrated.57. The genetically modified bacterial cell of any one of 38-56, whereinthe genetically modified bacterial cell is capable of treating ametabolic disease or disorder.58. The genetically modified bacterial cell of any one of 38-57, whereinthe genetically modified bacterial cell comprises one or moretherapeutic transgenes.59. The genetically modified bacterial cell of 58, wherein the one ormore therapeutic transgenes comprise one or more enzymes.60. A method comprising:

providing a bacterial cell; and

genetically modifying the bacterial cell to utilize as a carbon source arare carbohydrate of interest that is utilized as a carbon source byless than 50% (e.g., less than 30%, less than 10%, less than 3%, lessthan 1%, less than 0.3%, less than 0.1%, less than 0.03%, less than0.01%, less than 0.003%, less than 0.001%, less than 0.0001%, or none)of bacterial cells present in a human microbiome.

61. A method comprising:

providing a bacterial cell that utilizes as a carbon source a rarecarbohydrate of interest that is utilized as a carbon source by lessthan 50% (e.g., less than 30%, less than 10%, less than 3%, less than1%, less than 0.3%, less than 0.1%, less than 0.03%, less than 0.01%,less than 0.003%, less than 0.001%, less than 0.0001%, or none) ofbacterial cells present in a human microbiome; and genetically modifyingthe bacterial cell to express one or more therapeutic transgenes.

62. The method of 60 or 61, wherein the rare carbohydrate of interest isan isolated carbohydrate.63. The method of any one of 60-62, wherein the rare carbohydrate ofinterest is a polysaccharide.64. The method of any one of 60-63, wherein the rare carbohydrate ofinterest is a sulfated carbohydrate.65. The method of any one of 60-63, wherein the rare carbohydrate ofinterest is selected from the group consisting of porphyran, agarose,carrageenan, and any combination thereof.66. The method of any one of 60-65, wherein the rare carbohydrate ofinterest is a carbohydrate cleaved by a glycoside hydrolase belonging toglycoside hydrolase family GH86.67. The method of any one of 60-66, wherein the rare carbohydrate ofinterest is a sulfated polygalactan.68. The method of any one of 60-67, wherein the genetically modifiedbacterial cell is a genetically modified gut resident bacterial cell.69. The method of any one of 60-68, wherein the genetically modifiedbacterial cell is in the genus Bacteroides.70. The method of any one of 60-69, wherein the genetically modifiedbacterial cell is selected from B. acidifaciens, B. amylophilus, B.asaccharolyticus, B. barnesiae, B. bivius, B. buccae, B. buccalis, B.caccae, B. capillosus, B. capillus, B. cellulosilyticus, B.cellulosolvens, B. chinchilla, B. clarus, B. coagulans, B. coprocola, B.coprophilus, B. coprosuis, B. corporis, B. denticola, B. disiens, B.distasonis, B. dorei, B. eggerthii, B. endodontalis, B.faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. forsythus, B.fragilis, B. furcosus, B. galacturonicus, B. gallinarum, B. gingivalis,B. goldsteinii, B. gracilis, B. graminisolvens, B. helcogenes, B.heparinolyticus, B. hypermegas, B. intermedius, B. intestinalis, B.johnsonii, B. levvi, B. loescheii, B. macacae, B. massiliensis, B.melaninogenicus, B. merdae, B. microfusus, B. multiacidus, B. nodosus,B. nordii, B. ochraceus, B. oleiciplenus, B. oralis, B. oris, B.oulorum, B. ovatus, B. paurosaccharolyticus, B. pectinophilus, B.pentosaceus, B. plebeius, B. pneumosintes, B. polypragmatus, B.praeacutus, B. propionicifaciens, B. putredinis, B. pyogenes, B.reticulotermitis, B. rodentium, B. ruminicola, B. salanitronis, B.salivosus, B. salyersiae, B. sartorii, B. splanchnicus, B.stercorirosoris, B. stercoris, B. succinogenes, B. suis, B. tectus, B.termitidis, B. thetaiotaomicron, B. uniformis, B. ureolyticus, B.veroralis, B. vulgatus, B. xylanisolvens, B. xylanolyticus, B.zoogleoformans, and any combination thereof.71. The method of any one of 60-70, wherein the genetically modifiedbacterial cell comprises a carbohydrate-utilization gene or gene set.72. The method of 71, wherein the carbohydrate-utilization gene or geneset comprises one or more genes selected from the group consisting ofporphyranase, glycoside hydrolase, sulfatase, galactosidase, and anycombination thereof.73. The method of 71 or 72, wherein the carbohydrate-utilization gene orgene set comprises one or more nucleic acids encoding proteins that have80% or more sequence identity with BACPLE_1683-1706 from the B. plebeiusgenome.74. The method of any one of 71-73, wherein the carbohydrate-utilizationgene set comprises at least six genes.75. The method of any one of 71-74, wherein the carbohydrate of interestcannot be utilized as a carbon source by the genetically modifiedbacterial cell in the absence of the carbohydrate-utilization gene orgene set.76. The method of any one of 71-75, wherein the carbohydrate-utilizationgene or gene set is heterologous.77. The method of any one of 71-76, wherein the carbohydrate-utilizationgene or gene set is encoded on a plasmid, encoded on a bacterialartificial chromosome, or genomically integrated.78. The method of any one of 60-77, wherein the genetically modifiedbacterial cell is capable of treating a metabolic disease or disorder.79. The method of 61, wherein the one or more therapeutic transgenescomprise one or more enzymes.80. A method of facilitating colonization by a genetically modifiedbacterial cell, the method comprising:

(a) introducing a genetically modified bacterial cell into a gut of anindividual, wherein the genetically modified bacterial cell comprises aheterologous carbohydrate-utilization gene or gene set that provides thegenetically modified bacterial cell with the ability to use acarbohydrate of interest as a carbon source; and

(b) administering the carbohydrate of interest to the individual,thereby providing the genetically modified bacterial cell with thecarbon source.

81. The method of 80, wherein the genetically modified bacterial cell isa genetically modified gut resident bacterial cell.82. The method of 80 or 81, wherein the genetically modified bacterialcell is a Bacteroides cell.83. The method of 82, wherein the Bacteroides cell is a B. fragilis(Bf), B. distasonis (Bd), B. thetaiotaomicron (Bt), B. vulgatus (By), B.ovatus (Bo), B. eggerrthii (Be), B. merdae (Bm), B. stercoris (Bs), B.uniformis (Bu), or B. caccae (Bc) cell.84. The method of any one of 80-83, wherein the carbohydrate of interestcannot be utilized as a carbon source by the genetically modifiedbacterial cell in the absence of the heterologouscarbohydrate-utilization gene or gene set.85. The method of any one of 80-84, wherein the carbohydrate of interestis utilized as a carbon source by less than 50% (e.g., less than 30%,less than 10%, less than 3%, less than 1%, less than 0.3%, less than0.1%, less than 0.03%, less than 0.01%, less than 0.003%, less than0.001%, less than 0.0001%, or none) of other gut bacterial cells presentin the gut of the individual.86. The method of any one of 80-85, wherein the carbohydrate of interestis porphyran.87. The method of 86, wherein the heterologous carbohydrate-utilizationgene or gene set comprises one or more nucleic acids encoding theproteins that have 80% or more sequence identity with BACPLE_1683-1706from the B. plebeius genome.88. The method of any one of 80-87, wherein the genetically modifiedbacterial cell is a therapeutic bacterial cell.89. The method of any one of 80-88, wherein the genetically modifiedbacterial cell comprises a nucleic acid molecule comprising aheterologous nucleic acid sequence that encodes a therapeuticpolypeptide.90. A genetically modified bacterial cell, comprising:

a heterologous carbohydrate-utilization gene or gene set that providesthe genetically modified bacterial cell with the ability to use a rarecarbohydrate of interest as a carbon source.

91. The genetically modified bacterial cell of 90, wherein thegenetically modified bacterial cell is a genetically modified gutresident bacterial cell.92. The genetically modified bacterial cell of 90 or 91, wherein thegenetically modified bacterial cell is a Bacteroides cell.93. The genetically modified bacterial cell of 92, wherein theBacteroides cell is a B. fragilis (Bf), B. distasonis (Bd), B.thetaiotaomicron (Bt), B. vulgatus (By), B. ovatus (Bo), B. eggerrthii(Be), B. merdae (Bm), B. stercoris (Bs), B. uniformis (Bu), or B. caccae(Bc) cell.94. The genetically modified bacterial cell of any one of 90-93, whereinthe carbohydrate of interest cannot be utilized as a carbon source bythe genetically modified bacterial cell in the absence of theheterologous carbohydrate-utilization gene or gene set.95. The genetically modified bacterial cell of any one of 90-94, whereinthe carbohydrate of interest is porphyran.96. The genetically modified bacterial cell of 95, wherein theheterologous carbohydrate-utilization gene or gene set comprises one ormore nucleic acids encoding the proteins that have 80% or more sequenceidentity with BACPLE_1683-1706 from the B. plebeius genome.97. The genetically modified bacterial cell of any of 90-96, wherein thegenetically modified bacterial cell is a therapeutic bacterial cell.98. The genetically modified bacterial cell of any of 90-97, wherein thegenetically modified bacterial cell comprises a nucleic acid moleculecomprising a heterologous nucleic acid sequence that encodes atherapeutic polypeptide.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

To facilitate robust colonization, a privileged niche was created byintroducing a carbohydrate into the diet that can only be utilized bythe target bacteria. Remarkably, a single substrate was sufficient forboosting the colonization level of an introduced strain to more thanhalf of the total microbes in the gut of conventional mice, which areexpected to harbor hundreds of distinct bacterial strains. In additionto boosting total colonization levels, the data presented herein showthat this privileged niche results in more predictable levels of targetbacteria in different microbial communities. Finally, the data presentedherein demonstrate that the ability to utilize the carbohydrate can betransferred to a naïve species.

Results Selection of Polysaccharides Suitable for Establishing aPrivileged Niche

It was hypothesized that establishing a privileged niche that isinaccessible to other gut microbes would enable a predictably high levelof colonization in the gut by an introduced bacteria capable ofoccupying the privileged niche. Most substrates that are known to besafely consumable by humans and to be capable of supporting growth ofbacteria are readily consumed by the host or native bacterial species inthe gut and thus do not produce a privileged niche. Due to the widediversity in carbohydrate utilization capabilities amongst gut bacteria,carbohydrates were chosen as the substrate for creating a privilegedniche. It was reasoned that carbohydrates that are uncommon in the dietand are either rarely or not consumed by the gut bacteria in the humanpopulation of interest would be suitable substrates for creating aprivileged niche. The marine carbohydrates porphyran and agarose areboth known to be safe to consume yet are rare in Western diets. Theglycoside hydrolase family containing agarases and porphyranases, GH86,was found to be underrepresented in deeply-sequenced metagenomic stoolsamples from healthy adult Americans in the Human Microbiome Project(FIG. 1). Thus, these carbohydrates are likely to not be consumed bycommon American gut bacteria. In contrast to GH86, which was completelyabsent in 44% of samples and has a mean abundance of 0.00015, GH32,which hydrolyzes commonly consumed substrates such as the fructanpolysaccharide inulin, was present in all samples and had a meanabundance of 0.0049.

Isolation of Strains Capable of Consuming Privileged Substrates

To isolate a strain of gut bacteria from the environment that wascapable of growing on a carbohydrate of interest, a minimal media (seeSalyers et al., Appl. Environ. Microbiol. 33, 319-322 (1977)) wasdesigned for Bacteroides, the most abundant bacterial genus present inthe Western gut, in which the carbohydrate of interest was the solecarbon source. Strains capable of utilizing porphyran, NB001, (FIG. 2,panel A) or agarose, NB002 and NB003, were isolated from the environmentand the genomes of these strains were sequenced (Table 1). The porphyranutilizing strain NB001 contained a putative polysaccharide utilizationlocus (PUL) (SEQ ID NO: 67) on a mobile element that shares 71% identitywith a previously identified porphyran-utilizing strain (Hehemann etal., Nature 464, 908-912 (2010); Hehemann et al., Proc. Natl. Acad. Sci.U.S.A 109, 19786-19791 (2012)). The two agarose utilizers were found tohave a putative PUL (SEQ ID NO: 68 and 69) sharing over 99% identitybetween the two strains and include genes with high homology to agarasesof marine bacteria.

TABLE 1 Description of bacterial strains. Name Description SpeciesSource NB001 Porphyran consuming Bacteroides environmental Bacteroidesovatus isolate NB002 Agarose consuming Bacteroides environmentalBacteroides dorei isolate NB003 Agarose consuming Bacteroidesenvironmental Bacteroides uniformis isolate NB004 Porphyran naïveBacteroides environmental Bacteroides vulgatus isolate NB006 NB001 withBacteroides NB001 + pWW124 GFP/Erm marker ovatus (SEQ ID NO: 60) NB007Porphyran strain with Bacteroides NB001 + UV mut. tdk deletion ovatusNB008 Knockout of porphyran Bacteroides NB001 +/− pWD034 utilizationovatus (SEQ ID NO: 61) NB009 NB004 + Short Bacteroides NB004 + pWD037Porphyran PUL vulgatus (SEQ ID NO: 62) NB010 NB004 + Medium BacteroidesNB004 + pWD036 Porphyran PUL vulgatus (SEQ ID NO: 63)

Porphyran Establishes a Privileged Niche Resulting in RobustColonization

An in vitro experiment was performed to test the hypothesis thataddition of porphyran can increase the ability of an introducedporphyran utilizing bacteria to colonize a complex community at a high(and predictable) level. The strain NB006 was created by modifying NB001to contain an erythromycin resistance cassette expressing GFP tofacilitate monitoring abundance via plating. NB006 was added to thecomplex communities, which were then grown and serially passaged in richmedia either in the presence or absence of porphyran (FIG. 2, panel B).In the absence of porphyran, NB006 colonization levels reached widelyvariable abundances across the 8 complex communities, spanning a760-fold range by the seventh day. In the presence of porphyran, notonly did the abundance of NB006 increase for all eight communities, butthe deviation in abundance between the different communities alsodecreased considerably, spanning only a 3-fold range by the seventh day.

Next tested was the ability of porphyran to increase the abundance ofNB006 in mice harboring a conventional complex microbiota (FIG. 2). Onegroup of mice was given the standard (STD) rodent diet, while a secondgroup was fed a polysaccharide deficient (PD) diet. After three days oneach diet, both groups of mice received NB006. When both groups received1% porphyran in the drinking water, abundance of NB006 increased byalmost 3 logs in mice on the STD diet, and almost 4 logs in mice on thePD diet. Based on monitoring total anaerobic colony forming units, thisrepresents an increase in NB006 from roughly 0.1% of total anaerobic CFUin both groups, to approximately 25% of total in mice fed the STD dietand 48% in mice fed the PD diet.

Additionally tested was the ability of porphyran to allow NB006 tostably colonize a microbiota that resists colonization. Mice wereadministered NB008, an isogenic strain to NB006 that cannot utilizeporphyran, and this rendered them resistant to colonization by NB006 asthis strain was cleared from the mice within five days afterintroduction (FIG. 2, panel D). When mice were administered porphyranfor three days simultaneous to introduction of NB006, NB008 decreased inabundance by three logs while NB006 remained constant and highlyabundant (roughly 67% of total anaerobic CFU). After removal ofporphyran, NB008 increased back to pre-challenge levels while NB006decreased to one log lower abundance than NB008. After re-introductionof porphyran for seven days, NB006 displaced NB008 in a stable fashion,maintaining colonization of the mice in the absence of porphyran forthirty days. Additionally, NB008 was now excluded from the mice whenre-introduced (FIG. 2, panel E).

Transferring the Key Porphyran Utilization Genes is Sufficient forConferring Porphyran Utilization to a Naïve Species

To confirm that the mobile element of interest in NB001 (see SEQ ID NO:67) was necessary for the ability to grow on porphyran as a sole carbonsource, an 8-gene operon was deleted that contained key glycosidehydrolases predicted to act on porphyran and constituted 16% of themobile element. This deletion was sufficient to eliminate the ability togrow on porphyran (FIG. 3).

The porphyran mobile element from NB001 contains an approximately 30 kbdeletion relative to the homologous mobile element from B. plebeius,suggesting many of the genes in the region are likely not to be involvedin porphyran utilization (Figure. 4). To identify a minimal gene setnecessary for porphyran utilization, portions of the PUL weresynthesized of varying sizes and were integrated into the genome of aBacteroides strain not capable of growth on porphyran (NB004). Initiallyprotocols used previously to transfer a five gene PUL into a Bacteroideswere followed, but it was found that the much larger porphyran PUL (upto 34 genes and 60 kb) was not compatible with previously describedtechniques. Instead, the different sized porphyran PULs (FIG. 4) wereassembled into a plasmid with components necessary for yeast assembly,E. coli conjugation and Bacteroides genomic integration (FIG. 5), whichwas sufficient to integrate correctly assembled porphyran PULs intonaive Bacteroides. The shortest PUL candidate, containing 10 genesincluding both of the predicted porphyranases in the PUL, was notsufficient to impart porphyran utilization. However, when expanded to 24of the 34 total genes, integration of this medium-sized PUL wassufficient to confer growth on porphyran (FIG. 6).

Discussion

Using a privileged niche strategy, an unprecedentedly high colonizationlevel of an introduced strain was obtained in a complex community.Combining two or more privileged niches, potentially using dietarysubstrates from distinct geographic regions, could further broaden theeffectiveness of this strategy to allow robust colonization in the eventthat one of the two rare substrates is utilized by an individual'sendogenous microbiota. Marine carbohydrates (e.g. porphyran, agarose,agaropectin, carrageenan, and marine microbe exopolysaccharides) offerpromising substrates to establish a synthetic niche, as they are nottypically found in human diets and thus are less likely to be utilizedby human gut microbes. As demonstrated in this disclosure, marinebacteria possess utilization genes that can be transferred to gutadapted species. For example, we were able to demonstrate this with theporphyran PUL (SEQ ID NO: 67) and with putative agarose utilization PULs(SEQ ID NO: 68 and 69), which conferred growth on agarose when presentnatively or when transferred to a naïve strain. Marine carbohydrates aretypically easy to extract and generally safe to consume.

This strategy for facilitating robust colonization can facilitate arange of therapeutic applications where it is important to establish ahigh or a predictable number of natural or engineered therapeuticmicrobes. For example, it may be important to colonize the gut at a highenough level to ensure that therapeutic activities may overcomecompeting activities of other microbes. As an example, this may beimportant when attempting to, for instance, change the short chain fattyacid profile or reduce the accumulation of harmful chemicals that areproduced in the gut. Achieving predictable colonization can also beimportant for therapeutics that must be carefully dosed, such asbacteria with pro- or anti-inflammatory activities which could be usedas an adjuvant for cancer immunotherapy or in the treatment ofInflammatory Bowel Diseases. This method of ensuring robust colonizationcould also be used to displace a harmful species by establishing anon-harmful version that can effectively compete for limiting resourceswith the harmful strain.

Materials and Methods Identifying Rare Glycoside Hydrolase Subfamilies

87 fecal metagenomes sequenced by the HMP were downloaded andreprocessed (quality filtered and dynamically trimmed). Thesemetagenomic sequences were then translated to their respective codingsequences and Hidden Markov Models were applied to identify all CAZYmes.The data were normalized to the total count of assigned CAZYme codingsequences per sample.

Porphyran Extraction

Culinary Nori was added to ten times the weight of water and autoclavedfor approximately 3 hours. The autoclaved Nori was then strained toremove particulates and then diluted five fold into 100% ethanol andallowed to settle for six hours. This was then centrifuged for 15minutes at 30,000 g to pellet the precipitate, which was then dried at80° C.

Isolation of Porphyran and Agarose Utilizing Strains

A bacteroides minimal media was prepared as described by Salyers et al(see Salyers et al., Appl. Environ. Microbiol. 33, 319-322 (1977)) withaddition of 200 μg/ml Gentamycin and porphyran as 0.8% Nori extractadded as the sole carbon source. Primary sewage effluent was collected,settled for approximately two hours and diluted ten-fold into the media,which was then incubated anaerobically for 24 hours at 37° C. Theculture was then further diluted 200-fold into the fresh media andincubated another 24 hours anaerobically at 37° C. The saturated culturewas then plated as serial dilutions onto Blood-Heart-Infusion media+10%horse blood agar plates and incubated 24 hours anaerobically at 37° C.Colonies were then picked into fresh media, incubated 24 hoursanaerobically at 37° C. and prepared for analysis and cryogenic storage.

Assaying Abundance of Porphyran Utilizer in Complex Cultures

An erythromycin resistance cassette was genomically integrated intoNB001. The abundance of this strain, NB006, within each complexcommunity could then be easily assessed via selective plating. The eightcommunities and NB006 were separately grown in rich media, and thenNB006 was added to each community at a 1:20 dilution. The complexcommunities containing NB006 were subsequently grown and diluted 1:1000daily in rich media either with or without a supplementation of 0.4%Nori extract, a culturing strategy that mimics the requirement forongoing division necessary to sustain colonization of the gut. Todetermine the CFU/μL of NB006, 10 μL of each saturated culture wasdiluted into 200 μL of PBS, further successively diluted 1:10 in PBSseven times, 4 μL of each dilution was spotted onto a plate containingBrain Heart Infusion Blood agar with 200 μg/mL gentamycin and 10 μg/mLerythromycin, and after 24 hours of anaerobic growth at 37° C. colonycounts were enumerated. Both significantly increased abundance andreduced variance of NB006 across the eight communities was observed overthe course of a week.

Assaying Abundance of Porphyran Utilizer in Mice

To test ability of porphyran to toggle abundance of NB006 in vivo, onegroup of four male Restricted Flora Swiss-Webster mice (Taconic) weremaintained on standard rodent chow (STD diet), while an identical groupwas given a polysaccharide deficient diet (Bio-Serv, AIN-93G 68%glucose). Both groups were administered approximately 10{circumflex over( )}8 CFU of NB006. Abundance of NB006 in the feces was monitored byselective plating on Brain Heart Infusion Blood agar with 200 μg/mLgentamicin and 10 μg/mL erythromycin. Both groups received regular waterfor five days, 1% porphyran in the drinking water for five days, andthen were switched back to regular water for the duration of theexperiment.

To test ability of porphyran to allow for stable colonization of aporphyran utilizer into a resistant microbiota, two groups of three maleRestricted Flora Swiss-Webster mice (Taconic) were administered NB008,an isogenic strain to NB006 that cannot use porphyran. Abundance ofNB008 was monitored by introducing a tetracycline-resistance cassetteand selectively plating on Brain Heart Infusion Blood agar with 200μg/mL gentamicin and 2 μg/mL tetracycline. After one week, both groupsof mice received NB006 and one group received 1% porphyran in thedrinking water for three days, regular water for seven days, and then 1%porphyran in the drinking water for seven days, until being switchedback to and maintained on regular water for the duration of theexperiment. Abundance of NB006 was monitored by selectively plating asabove with erythromycin selection. Finally, NB008 was re-introduced tomice at day 48 and monitored in the feces.

Genome Sequencing and Analysis

Genomic DNA was isolated from Bacteroides strains capable of growth onporphyran (NB001) (SEQ ID NO: 67) or agarose (NB002, NB003) (SEQ ID NO:68 and 69, respectively) using a PureLink Genomic DNA Mini Kit(Invitrogen). Samples were prepared for multiplexed Illumina sequencingusing a Nextera XT DNA Library Preparation Kit (Illumina) and run on anIllumina MiSeq using a 2×150 bp paired-end kit. Approximately 10 millionsequencing reads were obtained for each sample. De novo assembly of thereads was performed with the Geneious De Novo Assembler (Biomatters),yielding an average coverage of ˜100 reads/bp. Gene annotation andalignment was also performed using Geneious.

Cloning and Transferring the Porphyran Utilization Genes.

Candidate porphyran PUL regions from the conserved mobile element ofNB001 were transferred to Bacteroides vulgatus (NB004) via a three-stepprocess, which is summarized here and expanded on in the sections below.First, the PUL genes of interest were amplified via PCR and assembledvia yeast assembly into a custom shuttle vector capable of propagationin S. cerevisiae and E. coli as well as conjugation and genomicintegration into Bacteroides species. Next, correctly assembled plasmidswere transferred from yeast to the S17-1 conjugation strain of E. coli(see Simon et al., Nat. Biotechnol. 1, 784-791 (1983)) byelectroporation. Finally, the plasmids were integrated into the genomeof B. vulgatus via conjugation and antibiotic selection, followed bysequence-verification using whole genome sequencing.

Design of Porphyran PUL Plasmids

A homolog (71% identity) of a porphyran mobile element was identified inthe NB001 genome using the Mauve genome aligner (see e.g., Hehemann etal., Proc. Natl. Acad. Sci. U.S.A. 109, 19786-19791 (2012)). Based ongene annotations and the sequence alignment between these two regions(FIG. 4), three candidate regions of varying sizes (20 kb, 40 kb, and 60kb) were selected that were predicted to potentially be sufficient toconfer growth on porphyran to a naive Bacteroides strain. The shortestof these candidate PULs contained homologs of BACPLE_1688-1699 from theB. plebeius genome, the medium-sized PUL contained homologs ofBACPLE_1683-1706, and the longest PUL contained homologs ofBACPLE_1669-1706 (see, e.g., Table 6).

Previously-described techniques for PUL transfer (Sonnenburg et al.,Cell 141, 1241-1252 (2010)) were insufficient to move PULs of this sizebetween species. Therefore, a novel PUL transfer strategy was designedthat utilized a custom shuttle vector containing a yeast selectablemarker and origin of replication, a bacterial artificial chromosomeorigin and selectable marker for plasmid-based propagation of large DNAsin E. coli, a conjugative origin of transfer to enable conjugation fromE. coli to Bacteroides species, as well as an integrase and selectablemarker to enable integration into the Bacteroides genome. This vectorwas divided into three precursor fragments with flanking homology suchthat only upon proper assembly would the vector be capable ofpropagation in yeast (Tables 3 and 4). The first fragment (Vector_01)was an AscI digestion of pWD011, which was custom built to contain allBacteroides-specific machinery as well all yeast vector components asidefrom a portion of the KanMX selectable maker. The remainder of the KanMXmarker was supplied via the PCR product Vector_02, which also served tobridge Vector_01 and Vector_03. Vector_03 was an AscI digest of pWD012,which was derived from a blunt religation of the commercially-availablepEZ-BAC vector (Lucigen). The PUL candidates were divided into multiple6 kb fragments to be used in a yeast assembly reaction (Tables 3 and 5),and PCR primers were designed such that neighboring fragments wouldshare ˜200 bps of homology (Table 2).

TABLE 2 List of oligonucleotides used to build the porphyran PUL plasmidSEQ  ID ID Sequence NO: AA34 GGGTACAGAAAATCTCGGTC 35 AA35TTCATCATGTCGTACGAAGG 36 AA36 TACTTCCATTTGGGGTGAAG 37 AA37TACAGTCCCTTTGGACAATG 38 AA38 CATACTTTAGCATCGTCGAAAAG 39 AA39TTTCCATTTCCAGGATTCCC 40 AA40 GTAGCTCCGGTGACATTTAC 41 AA41TCTTTAGCTGAAGAAACGGC 42 AA42 AAGCTTGCGTATGTCGATAG 43 AA43TTATCGCCATTCTTCAGCAG 44 AA44 TGGCATCCGACGAATATAAG 45 AA45TTTGGAATAGGCCAGTATGC 46 AA46 AGGTAAAGGCACTGTTTTCC 47 AA47ATATAGCCGGAGATTCTCCG 48 AA48 CATCTACATCATGTCGGACG 49 AA49CTGTCCGGTCATGATACATG 50 AA50 GATTCTCTTGGGGACAGAAC 51 AA51AGTTTCCCATTTCACGTCTG 52 AE30 ATTTATCTATCCATTCAGTTTGATTTCTCAGG 53ACTTTACATCGTCCTGAAAGTATTTGTTttttggg tgttgatatggcag AE31ATTTATCTATCCATTCAGTTTGATTTCTCAGG 54 ACTTTACATCGTCCTGAAAGTATTTGTTaatcccaatacagtctgttactg AE45 GTGAGTTGATTGCTACGTAAATAACTTCGTA 55TAGCATACATTATACGAAGTTATGGACTAcgc aggtcaatatccggaa AE46GTGAGTTGATTGCTACGTAAATAACTTCGTA 56 TAGCATACATTATACGAAGTTATGGACTAggtaaacctccccgatgg AE47 GTGAGTTGATTGCTACGTAAATAACTTCGTA 57TAGCATACATTATACGAAGTTATGGACTAtttag aacatatttttccgatttgccag AE49AAACAGCATTCCAGGTATTAGAAG 58 AE50 CACTGCCCGCTTTCCAGTCGGGAAACCTGT 59GCGGCCGCTTTCCTTCTTTCTCTCTTCTGGc agtatagcgaccagcattc

TABLE 3 List of PCR products used to build the porphyran PUL plasmids.SEQ Length Prim- Prim- ID ID (bps) er 1 er 2 Template NO: PCR_01 6,220AA35 AA36 NB001 Genomic DNA 71 PCR_02 6,280 AA37 AA38 NB001 Genomic DNA72 PCR_03 6,247 AA39 AA40 NB001 Genomic DNA 73 PCR_04 6,169 AA41 AA42NB001 Genomic DNA 74 PCR_05 6,232 AA43 AA44 NB001 Genomic DNA 75 PCR_066,187 AA45 AA46 NB001 Genomic DNA 76 PCR_07 6,185 AA47 AA48 NB001Genomic DNA 77 PCR_08 6,178 AA49 AA50 NB001 Genomic DNA 78 PCR_09 6,162AE45 AA34 NB001 Genomic DNA 79 PCR_10 6,138 AA51 AE30 NB001 Genomic DNA80 PCR_11 5,359 AE46 AA40 NB001 Genomic DNA 81 PCR_12 4,638 AE47 AA42NB001 Genomic DNA 82 PCR_13 3,524 AA47 AE31 NB001 Genomic DNA 83Vector_02 769 AE49 AE50 KanMX 84 (SEQ ID NO: 70)

TABLE 4 List of digests used to build the porphyran PUL plasmids. LengthRestriction ID (bps) Plasmid Enzyme Vector_01 4,730 pWD011 (SEQ ID NO:64) Ascl Vector_03 7,234 pWD012 (pEZ-BAC) Ascl (SEQ ID NO: 65)

TABLE 5 List of porphyran PUL plasmids. Plasmid Length Name Description(bps) Parts used in yeast assembly pWD035 Long 72,558 PCR_01, PCR_02,PCR_03, (SEQ ID Porphyran PCR_04, PCR_05, PCR_06, NO: 66) PUL PCR_07,PCR_08, PCR_09, PCR_10, Vector_01, Vector_02, Vector_03 pWD036 Medium53,695 PCR_04, PCR_05, PCR_06, (SEQ ID Porphyran PCR_07, PCR_08, PCR_10,NO: 63) PUL PCR_11, Vector_01, Vector_02, Vector_03 pWD037 Short 32,364PCR_05, PCR_06, PCR_12, (SEQ ID Porphyran PCR_13, Vector_01, NO: 62) PULVector_02, Vector_03

Yeast Assembly of Porphyran PUL Plasmids

All fragments were prepared for yeast assembly via PCR with NEB Q5polymerase (Table 3) or restriction digest with AscI from NEB (Table 4).Cleanup reactions were performed with a Zymo Clean and Concentrator kit,and the resulting DNA concentrations were measured using a Nanodrop 1000(Thermo Scientific). Yeast assembly of plasmids pWD035-37 (Table 5) wascarried as described (see, e.g., Chandran et al., Methods Mol. Biol.1472, 187-192 (2017)), using 100 fmols of each part in a lithium acetatetransformation of the S. cerevisiae strain BY4741. After rescuing thetransformed cells for 1 hour at 30° C. in YPD media, cells were platedon YPD plates supplemented with G418 (200 μg/ml). After incubation for48 hours at 30° C., colonies were grown up in YPD+G418 and screened forsuccessful plasmid assembly using colony PCR. Approximately 90% ofscreened clones appeared to contain a correctly assembled plasmid.

Transfer of Porphyran PUL Plasmids from Yeast to E. coli

Yeast strains harboring properly assembled plasmids were back diluted1:100 into YPD+G418 and grown at 30° C. for 6 hours to an OD of 2.500 μlof each culture was transferred to a microcentrifuge tube, centrifuged,and resuspended in 100 μl of PBS buffer. The cell mixture was thenvortexed alongside ˜50 0.5 mm glass disruptor beads (USA Scientific) for5 minutes to lyse the cells. 5 μl of the lysate was used in anelectroporation of 100 μl of electrocompetent S17-1 E. coli.Transformations were rescued for 1 hour in LB media with shaking at 37°C. and then plated onto agar plates with LB+ampicillin (100 μg/ml). Forlarge plasmids, fewer than 20 colonies were often obtained from a singleelectroporation. Colonies were grown up in LB+ampicillin and screenedfor successful plasmid transfer via colony PCR.

Conjugation of Porphyran PUL Plasmids into B. vulgatus

The PUL plasmids were then transferred from S17-1 E. coli into thegenome of an environmental isolate of B. vulgatus (NB004). It wasobserved that larger plasmids resulted in fewer conjugants onerythromycin-selective plates, with the largest plasmid (pWD035) notyielding any successful plasmid integrants. Despite this size bias, bothpWD036 and pWD037 were successfully integrated into NB004 as verified bywhole genome sequencing of the resulting strains.

In Vitro Growth Assays

Strains were grown overnight anaerobically at 37° C. in a tryptone-yeastextract-glucose (TYG) medium to saturation. Saturated cultures werediluted 1:50 into Salyer's Minimal Media supplemented with either 0.2%glucose or 0.8% porphyran extract. The diluted cultures were grownanaerobically at 37° C. for 12 hours, and 100 μL of each culture wastransferred to a 96-well microtiter plate. The optical density at 600 nmof each culture was measured using a Wallac Victor 2 1420 MultilabelCounter. OD600 readings were normalized by subtracting out thebackground detected when media lacking cells was measured.

Example 2 Synthetic Niche Generation Enables Strain Integration in theGut Microbiota

The dense microbial ecosystem in the gut is intimately connected tonumerous facets of human biology, and manipulation of the gut microbiotahas broad implications for human health. In the absence of profoundperturbation, the abundance of bacterial strains that reside within anindividual is largely stable over time. In contrast, the fate ofexogenous commensal and probiotic strains applied to an establishedmicrobiota is variable, largely unpredictable, and greatly influenced bythe background microbiota.

In this example, a synthetic metabolic niche was generated viaadministration of a privileged nutrient source, and reliable integrationof an exogenous Bacteroides strain at predictable abundances into miceharboring diverse communities of gut microbes was demonstrated. Dietarymarine polysaccharides not accessible to other members of the gutcommunity, but utilized by the introduced strain facilitated predictableengraftment and boosted abundance by more than three orders ofmagnitude, independent of background microbiota. This targeted dietarysupport was sufficient to overcome priority exclusion by an isogenicstrain, and facilitated strain replacement. Transfer of this privilegednutrient-utilization system into a naïve strain of Bacteroides isdemonstrated, and finely tuned control of strain abundance in the gut byvarying polysaccharide input is shown. The data presented here highlightthe influence of nutrient availability in shaping microbiota membership,expand the ability to perform a broad spectrum of methods in the contextof a complex microbiota, and facilitate cell-based therapeuticstrategies in the gut.

Results and Discussion

Changes to the microbial membership of the highly competitive anddynamic gut microbiota can impact numerous aspects of host biology.Despite the importance of gut microbe composition in human health, therules governing invasion of commensal strains into an existing complexcommunity are not well understood. Resident strains often appear toexclude similar invading strains although in some cases, the opposite istrue: the niche occupied by existing strains can be exploited by asimilar invading strain. The inability to predict or control the outcomeof fecal microbiota transplants illustrates the need for basic insightinto the factors that influence whether new strains of bacteria canintegrate into a pre-existing, complex microbiota.

To characterize the extent to which incoming exogenous bacteria variablycolonize hosts with distinct microbiotas, mice harboring a conventionalmouse microbiota were used as were two groups of ex-germ free mice, eachcolonized with the gut microbiota from two different healthy humandonors from the United States (“humanized”) as model hosts. These threegroups of mice were administered a strain of Bacteroides ovatus (NB001),a prominent gut commensal species, which was modified genetically,adding erythromycin resistance and GFP-expression cassettes for trackingpurposes. NB001 was monitored by colony forming units (c.f.u.) in fecesvia selective plating, and colonies were verified by GFP-fluorescence(see Methods) over the course of seven days (FIG. 7a ). The differentcommunities (FIG. 10) varied in their ability to integrate NB001 (FIG.7b ), and one human microbiota was resistant to colonization, with allmice in the group exhibiting decreased abundance of the challenge strainover the seven day period.

Inulin was then administered to the three aforementioned groups of miceseven days after inoculation with NB001, which grows at a similar ratein vitro in a minimal medium containing either glucose or inulin as thesole carbohydrates (MM-glucose doubling time=157 min; MM-inulin doublingtime=127 min; FIG. 11b ). Over seven days of feeding an inulin-baseddiet (10% inulin w/v) to these three groups of mice, NB001 exhibitedhighly variable responses across the background microbiotas (range ofmean c.f.u. per ml in feces <4*10E4 to 2*10E10; FIG. 7c ). Thisvariability may be due to several factors that differentiate the threemicrobiotas including the varying degrees of competition from othercommunity members for this MAC, common in the Western diet.

In contrast, microbiota utilization of the non-ubiquitous seaweedpolysaccharide porphyran, found in the seaweed Porphyra yezoensis usedto prepare culinary nori, is much less common in US microbiotas. Theconsumption of porphyran by some Bacteroides species can be facilitatedby a horizontally-transferred PUL, which originated in marine bacteria.We hypothesized that this exotic dietary MAC would create a privilegedniche within the gut and promote integration of an exogenous straincompetent in its use. NB001 was capable of growth on porphyran in vitro(doubling time=98 min; FIG. 11c ), and so a custom nori diet (10% driedPorphyra yezoensis w/v) was administered to the three groups of miceseven days after inoculation with NB001. Indeed, in response to nori inthe diet a robust increase in abundance of NB001 was observed,irrespective of background microbiota. Additionally, the variability incolonization levels across communities was eliminated (range of meanc.f.u. per ml in feces 3*10E10 to 6*10E10; FIG. 7d ), indicatingspecificity of an exotic nutrient source and cognate utilization systemto promote bacterial growth in vivo. Access to nori substrates rescuedNB001 from below the limits of detection in the most resistantmicrobiota and boosted its abundance to levels indistinguishable fromthose achieved in the other two microbiotas (FIG. 7d ). Together thesedata suggest a powerful approach to reproducibly control strainintegration independent of the background microbiota, and implicatenutrient availability as a key modulator of strain integration into thegut community.

Given the context independence of strain integration via access to noripolysaccharides, it was next tested whether the population size could betoggled by addition and removal of the substrate in vivo. Conventionalmice were colonized with NB001 and c.f.u. in feces was tracked. The micewere switched in five day intervals between regular water, water with 1%porphyran extract, and a regular water washout. To test the influence ofcompeting polysaccharides in the diet, this experiment was performed onboth standard, MAC-rich chow and a MAC-deficient chow that provides noexogenous polysaccharides to the microbiota. In both dietary contexts,with or without other competing dietary MACs, NB001 responded robustlyto introduction of marine polysaccharides, showing a large and highlyreproducible increase in abundance of four orders of magnitude in theabsence of dietary MACs (FIG. 8a ) and three orders of magnitude in thepresence of diverse dietary MACs (FIG. 8b ). This response wascontingent upon access to porphyran, as deletion of eight genes requiredfor its metabolism abolished the effect (FIG. 8c , FIG. 9a, 9b ).Additionally, the extract alone did not significantly affect thecomposition of the background microbiota (FIG. 12), further supportingthe lack of porphyran use by members of the background community.

Bacteroides species engage in interesting colonization behavior in whichan early colonizer will exclude a challenging isogenic strain, thephenomenon known as priority effects in the ecological literature. Wehypothesized that this behavior, though previously demonstrated ingnotobiotic mice, would also occur in the context of a complexcommunity, and that it could be overcome by providing a privilegednutrient to the challenging strain to create a distinct metabolic niche.Indeed, an early-colonizing NB001 strain excluded an NB001 challengestrain in conventional mice (FIG. 8d ). Supplementing porphyran in thediet, accessible only to the challenging strain, allowed the challengingstrain to overcome the priority effect, and resulted in displacement ofthe early colonizer by the challenging strain (FIG. 8e ). Replacement ofthe early-colonizing strain required that the challenging strain haveaccess to porphyran (FIG. 13), and displacement was robust to subsequentchallenge by the original colonizer (FIG. 13). Interestingly, whenporphyran was supplemented for shorter periods of time, replacement ofthe original strain was incomplete: the early-colonizer recovered frombelow the levels of detection and co-existed with the secondarycolonizer after cessation of porphyran administration (FIG. 8f ).

The genome of NB001 was sequenced and the polysaccharide utilizationlocus (PUL) was identified (FIG. 9a ). Three different length PULs wereconstructed, ranging from 10 to 34 of the genes (20-60 kb) from thefull-length PUL (FIG. 9a ). Previous methods to transfer a five gene PULwere not sufficient for these much larger constructs, and so theconstructs were assembled in yeast before transferring to conjugative E.coli, and the constructs were integrated into the chromosome of twotarget strains unable to utilize porphyran for growth, Bacteroidesstercoris and Bacteroides thetaiotaomicron. Transfer of the shortest PULwas insufficient to impart growth on porphyran to either naïve speciesof Bacteroides tested (FIG. 9b ), but the medium PUL allowed for growthin vitro (FIG. 9b ). A significant drop-off in conjugation efficiencywas observed with the long PUL, and only B. stercoris yieldedtransconjugants, suggesting inter-species differences in amenability toaccepting large pieces of foreign DNA. When colonized into conventionalmice, abundance of B. thetaiotaomicron harboring the medium length PULcould be toggled up and down through addition of porphyran extract inthe water (FIG. 9c ).

Finally, the tunability of strain abundance was assessed by varying theporphyran supplemented in diet. Conventional mice were colonized with aGFP fluorescent NB001 strain and c.f.u. in the feces was tracked forfive days. 1%, 0.1%, or 0.01% weight by volume porphyran extract wasthen administered in the drinking water for five days, followed by asubsequent five-day washout with regular water. A ten-fold decrease inabundance of NB001 in the feces was observed with each ten-fold dilutionof extract administered in the water (FIG. 9d ), indicating fine controlover strain abundance through modulation of the porphyran concentration.To visually assess the spatial distribution and abundance of NB001 invivo in the presence of different amounts of porphyran, frozen tissuesections were prepared and endogenous GFP of the NB001 strain was imagedvia confocal microscopy. A substantial increase in GFP-positive cellswas seen when comparing mice given 0.01% vs. 1% extract (FIG. 9e ).

Predicting amenability of a given community to the introduction of a newstrain is a challenge given the diverse and divergent features ofdifferent individuals' pre-existing microbiotas, including whether ornot it is already inhabited by a strain capable of excluding the onebeing introduced. The results presented in this example demonstrate thatprivileged nutrient sources such as porphyran can modify resourcepartitioning in the gut microbiota, and systems such as those presentedhere can be deployed to enhance human health.

Methods Bacterial Culture and Strain Isolation

All bacterial growth was performed at 37° C. under anaerobic conditions.Growth for introduction into mice was performed in rich media(tryptone-yeast-glucose) with no antibiotics added. Growth curves andselective growth on nori extract were performed in Salyers Minimal Media(SMM 100 mL in dH₂O: 0.1 g (NH₄)₂SO₄, 0.1 g Na₂CO₃, 0.05 g L-cysteine,10 mL 1M KPO₄ pH7.2, 5 mL Mineral Salts (1 L in dH₂O: 18 g NaCl, 0.53 gCaCl₂*2H₂O, 0.4 g MgCl₂*6H₂O, 0.2 g MnCl₂*4H₂O, 0.2 g CoCl₂*6H₂O), 1 mL0.4 mg/mL FeSO₄, 0.1 mL 1 mg/mL Vitamin K₃, 0.1 mL Histidine/Hematin*,0.05 mL 0.01 mg/mL Vitamin B₁₂) with carbon source added to a finalconcentration of either 0.2% (FIG. 9b , glucose), 0.5% (FIG. 11), or0.8% (FIG. 9b , nori extract) weight by volume and filter sterilized.SMM was either made fresh the day of the experiment, or prepared withouthistidine/hematin or L-cysteine and stored at 4° C. for up to fourweeks, with the missing components added the day of the experiment.

C.f.u. were cultivated on brain-heart infusion blood agar (BHI-BA) withappropriate selective antibiotics (200 μg/mL gentamycin, and 25 μg/mLerythromycin or 2 μg/mL tetracycline), or on SMM agar plates (for GFPvisualization, 2×SMM tempered to 50° C.+ equal volume 3% agar temperedto 50° C.) with selective antibiotics.

NB001 (porphyran MAC utilizing B. ovatus) and NB004 (naïve B. stercoris)were isolated from primary waste effluent at the San Jose WastewaterTreatment Facility via selection in liquid culture (SMM) with 200 ug/mLgentamycin (as Bacteroides are naturally gentamycin resistant*), and forNB001, growth on 0.8% nori extract. Briefly, settled primary effluentwas diluted ten-fold into SMM and grown as above for 24 h, subculturedat 1:200 into fresh media and grown for 24 h, and plated in serialdilutions onto BHI-BA. Single colonies were picked into SMM for growthconfirmation, cryogenic storage, and downstream analysis. AGFP-expressing, erythromycin-resistant variant of NB001 was generated asdescribed previously.

Genome Sequencing and Analysis

Genomic DNA was isolated from NB001 and NB004 using a PureLink GenomicDNA Mini Kit (Invitrogen). Samples were prepared for multiplexedIllumina sequencing using a Nextera XT DNA Library Preparation Kit(Illumina) and run on an Illumina MiSeq using a 2×150 bp paired-end kit.Approximately 10 million sequencing reads were obtained for each sample.De novo assembly of the reads was performed with the Geneious De NovoAssembler (Biomatters), yielding an average coverage of ˜100 reads/bp.Gene annotation and alignment was also performed using Geneious.

Porphyran PUL Transfer and Knockout

Generation of the porphyran utilization deficient mutant was performedusing tdk counterselection as described previously. A thymidine kinase(tdk) deficient mutant (NB007) of NB001 was generated by exposing NB001to ultraviolet light for 60 seconds and plating on BHI-BA supplementedwith 200 ug/mL of 5-fluoro-2′-deoxyuridine (FUdR). Eight genes predictedto be essential for growth on porphyran (homologous to BACPLE_1692-1699)were knocked out using pWD034 (Table 7), a plasmid with 1.5 kb ofhomology upstream and downstream of the target region assembled viaGolden Gate Assembly into an erythromycin-resistant, tdk-containingvector.

TABLE 7 List of plasmids used in Example 2 Plasmid Name DescriptionLength (bps) Parts used in yeast assembly Relevant FIG. pWW3452 HighGFP, 6,031 N/A FIG. 7, erythromycin FIG. 9e resistance pNBU2-Erythromycin 4,909 N/A FIG. 2, ermGb resistance FIG. 12 pNBU2-Tetracycline 6,662 N/A FIG. 8, tetQb resistance FIG. 12 pWD034 PorphyranPUL 7,148 N/A FIG. 8c Deletion (main operon) pWD035 Long Porphyran72,558 PCR_01, PCR_02, PCR_03, FIG. PUL PCR_04, PCR_05, 9a, b PCR_06,PCR_07, PCR_08, PCR_09, PCR_10, Vector_01, Vector_02, Vector_03 pWD036Medium Porphyran 53,695 PCR_04, PCR_05, PCR_06, FIG. PUL PCR_07, PCR_08,9a, b, c PCR_10, PCR_11, Vector_01, Vector_02, Vector_03 pWD037 ShortPorphyran 32,364 PCR_05, PCR_06, PCR_12, FIG. PUL PCR_13, Vector_01, 9a,b Vector_02, Vector_03

Generation of the porphyran PUL knock-in strains required expansion ofprevious knock-in methods due to the large size of the PULs (20-60 kb).Based on gene annotations and sequence alignment to a previouslypublished mobile element conferring porphyran polysaccharide utilizationcapabilities, we designed three minimal PULs of varying sizes (20 kb, 40kb, and 60 kb, FIG. 9a ). To propagate such large pieces of DNA andintegrate them into the Bacteroides genome we used a three-step process:performing yeast assembly into a custom shuttle vector, propagating itin S. cerevisiae and E. coli, and then performing conjugation andgenomic integration into Bacteroides species. The minimal PULs were eachdivided into multiple 6 kb fragments with 200 bp homology between piecesand assembled in yeast with fragments containing the KanMX selectablemarker and CEN6/ARS4 origin for selection and growth in yeast, thebacterial artificial chromosome origin and chloramphenicol selectablemarker (from pEZ-BAC vector, Lucigen) for selection and growth in E.coli, and the conjugative origin and parts for integration and selectionin Bacteroides (Tables 2, 3, 4—see above). Yeast cells with successfullyassembled constructs were lysed by mechanical disruption with 0.5 mmglass disruptor beads (USA Scientific), and lysates mixed 1:20 withelectrocompetent E. coli S17-1 cells and electroporated. Successfullytransformed E. coli were then grown and conjugated with NB004 and B.thetaiotaomicron VPI-5482 (Bt) as previously described. NB004successfully integrated all PULs, but Bt was initially unable tointegrate any PUL constructs. To improve rates of genomic integration,we pre-integrated an NBU2 integrase-expressing plasmid with tetracyclineresistance into Bt, and this improved efficiency such that Bt conjugantswere obtained for both the short and medium length PUL constructs.

Nori Extract Preparation

Raw culinary nori derived from Porphyra yezoensis(https://www.rawnori.com) was added at 10% weight by volume to distilledwater and subjected to hot water extraction by autoclaving for threehours. The mixture was then cooled and centrifuged at 11,000×g.Resulting supernatant was ethanol precipitated by combining with200-proof ethanol to a final concentration of 80% ethanol, 20%supernatant and incubating at 4° C. for 24-72 h. Precipitate wasrecovered by centrifugation at 26,000×g and dried for 24 h before manualgrinding to generate a measurable powder.

Mice

Ex-germ free or restricted flora (RF) conventional Swiss-Webster mice(Taconic) were housed in gnotobiotic isolators and fed either anautoclaved standard diet (LabDiet 5K67) or a custom diet as indicatedbelow (Bio-Serv) in strict accordance with a Protocol for Care and Useof Laboratory Animals approved by the Stanford University AdministrativePanel of Laboratory Animal Care. Introduction of all Bacteroides strainsinto either ex-germ free or RF mice was performed by oral gavage of 10⁸c.f.u. of the given strain in culture media.

Mice were humanized (FIG. 7) with fecal samples from healthy humandonors were stored at −80° C., thawed and resuspended in pre-reduced PBSin anaerobic conditions at a 1:1 dilution, and 0.2 mL gavaged orallyinto germ free mice. The mice were allowed to equilibrate the humanmicrobiota for four weeks while consuming the standard lab diet. Oneweek before introduction of NB001, mice were switched to MAC-deficientchow (AIN-93G, 68% glucose). Seven days after introduction of NB001,mice were switched to custom diets with inulin or nori as the onlyavailable MACs (AIN-93G, 10% unique polysaccharide, 58% glucose). RFmice (FIG. 8, FIG. 9) were administered nori extract in the water at thepercent indicated, weight by volume.

16S rRNA Analysis

DNA was extracted from fecal samples using the PowerSoil 96-htp kit(MoBio), and amplified at the 16S v4 region (515F, 806R). Qiime 1.9 wasused to analyze the resulting Illumina-generated sequencing reads aspreviously described*. Data were rarified to the sample with the lowestnumber of reads (16384), and open-reference OTU picking via UCLUST andtaxonomy assignment through the Greengenes 13.8 database was performed.

Microscopy

Tissue was harvested and immediately fixed in 4% paraformaldehyde in PBSfor 48 hours at 4 C. Cassettes were transferred to 20% sucrose for 24 h,and then samples were embedded in OCT Compound (Tissue-Tek) beforesectioning at 30 μm on a Leica CM3050 S cryostat. Sections were stainedfor 30 minutes with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI;Sigma-Aldrich) and Alexa Fluor 594 Phalloidin (Life Technologies).Images were taken on a Zeiss LSM 700 confocal microscope.

Example 3

FIG. 14 (panels A-C). Access to porphyran allowed for crypt invasion andstable maintenance in the presence of an exclusionary primary colonizingstrain. Germ-free mice were colonized with B. thetaiotaomicron strainVPI-5482 that was unable to utilize porphyran. One week later, the micewere challenged with an isogenic strain harboring the medium-lengthporphyran PUL, and continued drinking normal water (panel A) or wereadministered 1% porphyran in the drinking water for four days (panel B,brackets). After four days, mice in panel B were switched to regularwater, and two mice were sacrificed and images of the proximal colonwere captured, demonstrating the challenge strain (panel C, green)colonizing the colonic crypts with the primary strain (panel C, red).Colony forming units were tracked in the feces for the duration of theexperiment, demonstrating that without access to 1% porphyran in thewater (panel A) the challenge strain clears, but with short-term accessthe challenge strain maintains stable co-colonization with the primarycolonizing strain. These data demonstrate that with access to porphyran,an incoming strain can enter the colonic crypts.

That which is claimed is:
 1. A method of colonizing the gut of a subjectwith a genetically modified, non-naturally occurring Bacteroides cell,the method comprising administering to the subject both: a) thegenetically modified Bacteroides cell, wherein the genetically modifiedBacteroides cell comprises a heterologous carbohydrate-utilization geneset that provides the genetically modified Bacteroides cell with anability to utilize as a carbon source a porphyran and comprises at leasttwelve genes and one or more nucleic acids encoding a porphyranase; andb) porphyran.
 2. The method of claim 1, wherein thecarbohydrate-utilization gene set comprises one or more nucleic acidsencoding a porphyranase from the B. plebeius or B. ovatus genome.
 3. Themethod of claim 1, wherein one of the nucleic acids encodes a proteinhaving 80% or more sequence identity to SEQ ID NO:
 19. 4. The method ofclaim 1, wherein one of the nucleic acids encodes a protein having 80%or more sequence identity to SEQ ID NO:
 21. 5. The method of claim 1,wherein the carbohydrate-utilization gene set comprises one or morenucleic acids each selected from the group consisting of nucleic acidsencoding a protein having 80% or more sequence identity to SEQ ID NOs:14-34.
 6. The method claim 1, wherein the carbohydrate-utilization geneset comprises one or more nucleic acids each selected from the groupconsisting of nucleic acids encoding a protein having 90% or moresequence identity to SEQ ID NOs: 14-34.
 7. The method of claim 1,wherein the carbohydrate-utilization gene set comprises one or morenucleic acids each selected from the group consisting of nucleic acidsencoding a protein having 95% or more sequence identity to SEQ ID NOs:14-34.
 8. The method of claim 1, wherein the genetically modifiedBacteroides cell further comprises one or more therapeutic transgenes.9. The method of claim 1, wherein the Bacteroides cell is not a B.plebeius cell or a B. ovatus cell.
 10. The method of claim 1, whereinthe carbohydrate-utilization gene set comprises at least twenty genes.11. The method of claim 5, wherein the carbohydrate-utilization gene setcomprises twelve or more nucleic acids each selected from the groupconsisting of nucleic acids encoding a protein having 80% or moresequence identity to SEQ ID NOs: 14-34.
 12. The method of claim 6,wherein the carbohydrate-utilization gene set comprises twelve or morenucleic acids each selected from the group consisting of nucleic acidsencoding a protein having 90% or more sequence identity to SEQ ID NOs:14-34.
 13. The method of claim 7, wherein the carbohydrate-utilizationgene set comprises twelve or more nucleic acids each selected from thegroup consisting of nucleic acids encoding a protein having 95% or moresequence identity to SEQ ID NOs: 14-34.
 14. The method of claim 1,wherein the carbohydrate-utilization gene set is at least 40 kb.
 15. Themethod of claim 1, wherein the carbohydrate-utilization gene set isencoded on a plasmid, encoded on a bacterial artificial chromosome, orartificially genomically integrated.
 16. The method of claim 15, whereinthe carbohydrate-utilization gene set is artificially genomicallyintegrated.
 17. The method of claim 1, wherein the genetically modifiedBacteroides cell and porphyran are administered orally.
 18. A method ofcolonizing the gut of a subject with a genetically modified,non-naturally occurring Bacteroides cell, the method comprising orallyadministering to the subject both: a) the genetically modifiedBacteroides cell, wherein the genetically modified Bacteroides cellcomprises a heterologous carbohydrate-utilization gene set that (i)provides the genetically modified Bacteroides cell with an ability toutilize as a carbon source a porphyran, (ii) comprises at least twentygenes, (iii) is at least 40 kb, (iv) comprises one or more nucleic acidsencoding a porphyranase, and (v) is encoded on a plasmid, encoded on abacterial artificial chromosome, or artificially genomically integrated;and b) porphyran.
 19. The method of claim 18, wherein the geneticallymodified Bacteroides cell further comprises one or more therapeutictransgenes.
 20. The method of claim 18, wherein the Bacteroides cell isnot a B. plebeius cell or a B. ovatus cell.
 21. The method of claim 18,wherein the carbohydrate-utilization gene set is artificiallygenomically integrated.
 22. The method of claim 18, wherein thecarbohydrate-utilization gene set comprises twelve or more nucleic acidseach selected from the group consisting of nucleic acids encoding aprotein having 80% or more sequence identity to SEQ ID NOs: 14-34. 23.The method of claim 18, wherein the carbohydrate-utilization gene setcomprises twelve or more nucleic acids each selected from the groupconsisting of nucleic acids encoding a protein having 90% or moresequence identity to SEQ ID NOs: 14-34.
 24. The method of claim 18,wherein the carbohydrate-utilization gene set comprises twelve or morenucleic acids each selected from the group consisting of nucleic acidsencoding a protein having 95% or more sequence identity to SEQ ID NOs:14-34.